Methods of using polynucleotide compositions

ABSTRACT

Compositions for stabilizing polynucleic acids and increasing the ability of polynucleic acids to cross cell membranes and act in the interior of a cell. In one aspect, the invention provides a polynucleotide complex between a polynucleotide and certain polyether block copolymers. The polynucleotide complex can further include a polycationic polymer, as well as suitable targeting molecules and surfactants. The invention also provides a polynucleotide complex between a polynucleotide and a block copolymer comprising a polyether block and a polycation block.

This is a division of Ser. No. 09/124,943 filed Jul. 30, 1998, now U.S.Pat. No. 6,221,959, which in turn is continuation-in-part of Ser. No.08/912,968, filed Aug. 1, 1997 now U.S. Pat. No. 6,353,055, which inturn is a continuation-in-part of Ser. No. 08/342,209, filed Nov. 18,1994, now U.S. Pat. No. 5,656,611.

FIELD OF THE INVENTION

The present invention relates to compositions of poly(nucleic acid)polymers such as RNA or DNA polymers and polycations that areassociated, either covalently or noncovalently, with block copolymers ofalkylethers. The complexes are well suited for use as vehicles fordelivering nucleic acid into cells.

BACKGROUND OF THE INVENTION

The use of antisense poly(nucleic acids) to treat genetic diseases, cellmutations (including cancer causing or enhancing mutations) and viralinfections has gained widespread attention. This treatment tool isbelieved to operate, in one aspect, by binding to “sense” strands ofmRNA encoding a protein believed to be involved in causing the diseasestate sought to be treated, thereby stopping or inhibiting thetranslation of the mRNA into the unwanted protein. In another aspect,genomic DNA is targeted for binding by the antisense polynucleotide(forming a triple helix), for instance, to inhibit transcription. SeeHelene, Anti-Cancer Drug Design, 6:569 (1991). Once the sequence of themRNA sought to be bound is known, an antisense molecule can be designedthat binds the sense strand by the Watson-Crick base-pairing rules,forming a duplex structure analogous to the DNA double helix. GeneRegulation: Biology of Antisense RNA and DNA, Erikson and Ixzant, eds.,Raven Press, New York, 1991; Helene, Anti-Cancer Drug Design, 6:569(1991); Crooke, Anti-Cancer Drug Design, 6:609 (1991). A serious barrierto fully exploiting this technology is the problem of efficientlyintroducing into cells a sufficient number of antisense molecules toeffectively interfere with the translation of the targeted mRNA or thefunction of DNA.

One method that has been employed to overcome this problem is tocovalently modify the 5′ or the 3′ end of the antisense polynucleic acidmolecule with hydrophobic substituents. These modified nucleic acidsgenerally gain access to the cells interior with greater efficiency.See, for example, Kabanov et al., FEBS Lett., 259:327 (1990); Boutorinet al., FEBS Lett., 23:1382-1390, 1989; Shea et al, Nucleic Acids Res.,18:3777-3783 (1990). Additionally, the phosphate backbone of theantisense molecules has been modified to remove the negative charge (seefor example, Agris et al., Biochemistry, 25:6268 (1986); Cazenave andHelene in Antisense Nucleic Acids and Proteins. Fundamentals andApplications, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker,New York, (1991) or the purine or pyrimidine bases have been modified(see, for example, Antisense Nucleic Acids and Proteins: Fundamentalsand Applications, Mol and Van der Krol, eds., p. 47 et seq., MarcelDekker, New York (1991); Milligan et al. in Gene Therapy For NeoplasticDiseases, Huber and Laso, eds., p. 228 et seq., New York Academy ofSciences, New York (1994). Other attempts to overcome the cellpenetration barrier include incorporating the antisense poly(nucleicacid) sequence into an expression vector that can be inserted into thecell in low copy number, but which, when in the cell, can direct, thecellular machinery to synthesize more substantial amounts of antisensepolynucleic molecules. See, for example, Farhood et al., Ann. N.Y. AcadSci., 716:23 (1994). This strategy includes the use of recombinantviruses that have an expression site into which the antisense sequencehas been incorporated. See, e.g., Boris-Lawrie and Temin, Ann. N.Y.Acad. Sci., 716:59 (1994). Others have tried to increase membranepermeability by neutralizing the negative charges on antisense moleculesor other nucleic acid molecules with polycations. See, e.g. Kabanov etal., Soviet Scientific Reviews, Vol. 11, Part 2 (1992); 30 Kabanov etal., Bioconjugate Chemistry 4:448 (1993); Wu and Wu, Biochemistry,27:887-892 (1988); Behr et al., Proc. Natl. Acad Sci U.S.A. 86:6982-6986(1989). There have been problems with systemically administeringpoylnucleotides due to rapid clearance degradation and lowbioavailability. In some cases it would be desirable to targetpolynucleotide molecules to a specific site in the body to specifictarget cells. Also, due to poor or low transport across biologicalbarriers (such as the blood-brain to barrier) the transport ofpolynucleotides to targets across this barrier is decreased orimpossible. Additionally, the problems with low oral or rectalbioavailability dramatically hinders the administration of suchpolynucleotides (including oligonucleotides).

Of course, antisense polynucleic acid molecules are not the only type ofpolynucleic acid molecules that can usefully be made more permeable tocellular membranes. To make recombinant protein expression systems, theexpression-directing nucleic acid must be transported across themembrane and into the eukaryotic or prokaryotic cell that will producethe desired protein. For gene therapy, medical workers try toincorporate, into one or more cell types of an organism, a DNA vectorcapable of directing the synthesis of a protein missing from the cell oruseful to the cell or organism when expressed in greater amounts. Themethods for introducing DNA to cause a cell to produce a new protein,ribozyme or a greater amount of a protein or ribozyme are called“transfection” methods. See, generally, Neoplastic Diseases, Huber andLazo, eds., New York Academy of Science, New York (1994); Feigner, Adv.Drug Deliv. Rev., 5:163 (1990); McLachlin, et al., Progr. Nucl. AcidsRes. Mol. Biol., 38:91 (1990); Karlsson, S. Blood, 78:2481 (1991);Einerhand and Valerio, Curr. Top. Microbiol. Immunol, 177:217-235(1992); Makdisi et al., Prog. Liver Dis., 10:1 (1992); Litzinger andHuang, Biochim. Biophys. Acta, 11, 13:201 (1992); Morsy et al., JA.M.A.,270:2338 (1993); Dorudi et al., British J. Surgery, 80:566 (1993).

A number of the above-discussed methods of enhancing cell penetration byantisense nucleic acid are generally applicable methods of incorporatinga variety of poly(nucleic acids) into cells. Other general methodsinclude calcium phosphate precipitation of nucleic acid and incubationwith the target cells (Graham and Van der Eb, Virology, 52:456, 1983),co-incubation of nucleic acid, DEAE-dextran and cells (Sompayrac andDanna, Proc. Natl. Acad. Sci., 12:7575, 1981), electroporation of cellsin the presence of nucleic acid (Pofter et al., Proc. Natl. Acad. Sci.,81:7161-7165, 1984), incorporating nucleic acid into virus coats tocreate transfection vehicles (Gitman et al., Proc. Natl. Acad. Sci.U.S.A., 82:7309-7313, 1985) and incubating cells with nucleic acidincorporated into liposomes (Wang and Huang, Proc. Natl. Acad. Sci.,84:7851-7855, 1987).

Another problem in delivering nucleic acid to a cell is the extremesensitivity of nucleic acids, particularly ribonucleic acids, tonuclease activity. This problem has been particularly germane to effortsto use ribonucleic acids as anti-sense oligonucleotides. Accordingly,methods of protecting nucleic acid from nuclease activity are desirable.

SUMMARY OF THE INVENTION

The invention relates to compositions of poly(nucleic acid) polymerssuch as RNA or DNA polymers and polycations that are associated, eithercovalently or noncovalently, with block copolymers of alkylethers. In apreferred embodiment, the poly(nucleic acids) are complexed with apolycation. The nucleic acid is stabilized by the complex and, in thecomplex, has increased permeability across cell membranes. Accordingly,the complexes are well suited for use as vehicles for delivering nucleicacid into cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus relates to compositions of poly(nucleic acid)polymers such as RNA or DNA polymers, and polycations that areassociated (either covalently or noncovalently) with cationic blockcopolymers.

Structure of Block Copolymers

Block copolymers are most simply defined as conjugates of at least twodifferent polymer segments (Tirrel, M. In: Interactions of Surfactantswith Polymers and Proteins. Goddard E. D. and Ananthapadmanabhan, K. P.(eds.), CRC Press, Boca Raton, Ann Arbor, London, Tokyo, pp. 59-122,1992). Some block copolymer architectures are presented below.

The simplest block copolymer architecture contains two segments joinedat their termini to give an A-B type diblock. Consequent conjugation ofmore than two segments by their termini yields A-B-A type triblock,A-B-A-B- type multiblock, or even multisegment A-B-C- architectures. Ifa main chain in the block copolymer can be defined in which one orseveral repeating units are linked to different polymer segments, thenthe copolymer has a graft architecture of, e.g., an A(B)_(n) type. Morecomplex architectures include for example (AB)_(n) or A_(n)B_(m)starblocks which have more than two polymer segments linked to a singlecenter.

In accordance with the present invention, all of these architectures canbe useful for polynucleotide delivery, provided that they contain (a) atleast one polycationic segment that will bind a polynucleotide (“bindingsegment”) and (b) at least one water soluble segment that willsolubilize the complex formed between the block copolymer andpolynucleotide (“solubilizing segment”).

In accordance with the present invention, binding and solubilizingsegments can be, independently of each other, linear polymers, randomlybranched polymers, block copolymers, graft copolymers, star polymer,star block copolymer, dendrimers or have other architecture includingbut not limited to combinations of the above listed structures. For thepurposes of the current invention all these structures are collectivelycalled herein “block copolymers”.

The degree of polymerization of the binding and solubilizing segments isbetween about 3 and about 10,000. More preferably, the degree ofpolymerization is between about 5 and about 2,000, still morepreferably, between about 20 and about 1,000. The molecular weights ofthe binding and solubilizing segments is between about 600 and about500,000. More preferably, the molecular weights are between about 1000and about 100,000, still more preferably, between about 2000 and about50,000.

Formulas XVIII-XXIII of the instant invention are diblocks andtriblocks. At the same time, conjugation of polycation segments to theends of polyether of formula XVII yields starblocks (e.g., (ABC)₄ type).In addition, the polyspermine of examples 13-15 (below) are branched.Modification of such a polycation with poly(ethylene oxide) yields amixture of grafted block copolymers and starblocks.

In one embodiment, the poly(nucleic acids) are complexed with apolycation. The nucleic acid is stabilized by the complex and, in thecomplex, has increased permeability across cell membranes. Accordingly,the complexes are well suited for use as vehicles for delivering nucleicacid into cells.

In a preferred first embodiment, the block copolymer is selected fromthe group consisting of polymers of formulas:

wherein A and A′ are A-type linear polymeric segments, B and B′ areB-type linear polymeric segments, and R¹, R², R³ and R⁴ are either blockcopolymers of formulas (I), (III) or (III) or hydrogen and L is alinking group, with the proviso that no more than two of R¹, R², R³ orR⁴ are hydrogen. In another preferred first embodiment of the invention,the polynucleotide composition further comprises a polycationic polymercomprising a plurality of cationic repeating units.

The composition provides an efficient vehicle for introducingpolynucleotide into a cell. Accordingly, the invention also relates to amethod of inserting poly(nucleic acid) into cells utilizing the firstembodiment polynucleotide composition of the invention.

In a second embodiment, the invention provides a polynucleotidecomposition comprising:

(a) a polynucleotide or derivative thereof,

(b) a block copolymer having a polyether segment and a polycationsegment, wherein the polyether segment comprises at least an A-typesegment, and the polycation segment comprises a plurality of cationicrepeating units.

In a preferred second embodiment, the copolymer comprises a polymer offormulas:

wherein A, A′ and B are as described above, wherein R and R′ arepolymeric segments comprising a plurality of cationic repeating units,and wherein each cationic repeating unit in a segment may be the same ordifferent from another unit in the segment. The polymers of thisembodiment can be termed “polyether/polycation” polymers. The R and R′,segments can be termed “R-type” polymeric segments or blocks.

The polynucleotide composition of the second embodiment provides anefficient vehicle for introducing the polynucleotide into a cell.

Accordingly, the invention also relates to a method of insertingpoly(nucleic acid) into cells utilizing the second embodimentcomposition of the invention.

In a third embodiment, the invention provides a polynucleotidecomposition comprising a polynucleotide derivative comprising apolynucleotide segment and a polyether segment attached to one or bothof the polynucleotide 5′ and 3′ ends, wherein the polyether comprises anA-type polyether segment.

In a preferred third embodiment, the derivative comprises a blockcopolymer of formulas:

wherein pN represents a polynucleotide having 5′ to 3′ orientation, andA, A′ and B are polyether segments as described above. In anotherpreferred third embodiment, the polynucleotide complex comprises apolycationic polymer.

Polymers of formulas (I), (II), (III) or (IV) can also be mixed witheach other or can be mixed either additionally or alternatively with oneor more of the polymers of formula (V-a or b), (VI-a or b), (VII-a or b)and (VIII-a or b) and/or with polynucleotide derivatives of formulas(IX-a, b, c, or d), (X-a, b, c, d, e, or f, (XI), (XII) or (XIII) toprovide an efficient vehicle for delivering poly(nucleic acid) to theinterior of cells.

The polynucleotide composition of the third embodiment provides anefficient vehicle for introducing the polynucleotide into a cell.Accordingly, the invention also relates to a method of insertingpoly(nucleic acid) into cells utilizing the third embodiment compositionof the invention.

A fourth embodiment of the invention relates to a polyetherpolycationcopolymer comprising a polymer, a polyether segment and a polycationicsegment comprising a plurality of cationic repeating units of formula—NH—R⁰, wherein R⁰ is a straight chain aliphatic group of 2 to 6 carbonatoms, which may be substituted, wherein said polyether segmentscomprise at least one of an A-type of B-type segment. In a preferredfourth embodiment, the polycation polymer comprises a polymer accordingto formulas:

wherein A, A′ and B are as described above, wherein R and R′ arepolymeric segments comprising a plurality of cationic repeating units offormula —NH—R⁰—, wherein R⁰ is a straight chain aliphatic group havingfrom 2 to 6 carbon atoms, which may be substituted. Each —NH—R⁰—repeating unit in an R-type segment can be the same or different fromanother —NH—R⁰— repeating unit in the segment. A preferred fourthembodiment further comprises a polynucleotide or derivative.

In a fifth embodiment, the invention provides a polycationic polymercomprising a plurality of repeating units of formula:

wherein R⁸ is:

(1) —(CH₂)_(n)—CH(R¹³)—, wherein n is an integer from 0 to about 5, andR¹³ is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having 1-6carbon atoms, or (CH₂)_(m)R¹⁴; where m is an integer from 0 to about 12and R¹⁴ is a lipophilic substituent of 6 to 20 carbon atoms;

(2) a carbocyclic group having 3-8 ring carbon atoms, wherein the groupcan be for example, cycloalkyl or aromatic groups, and which can includealkyl having 1-6 carbon atoms, alkoxy having 1-6 carbon atoms,alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkylindependently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy,fluoro or chloro substituents; or (3) a heterocyclic group, having 3-8ring atoms, which can include heterocycloalkyl or heteroaromatic groups,which can include from 1 to 4 heteroatoms selected from the groupconsisting of oxygen, nitrogen, sulfur and mixtures thereto, and whichcan include alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbonatoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein eachalkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy,carboxy, fluoro or chloro substituents.

R⁹ is a straight chain aliphatic group of 1 to 12 carbon atoms, and R¹⁰,R¹¹ and R¹² are independently hydrogen, an alkyl group of 1-4 carbonatoms. R⁹ preferably comprises 2-10 carbon atoms, more preferably, 3-8.R¹⁴ preferably includes an intercalating group, which is preferably anacrydine or ethydium bromide group. The number of such repeating unitsin the polymer is preferably between about 3 and 50, more preferablybetween about 5 and 20. This, polymer structure can be incorporated intoother embodiments of the invention as an R-type segment or polycationicpolymer. The ends of this polymer can be modified with a lipidsubstituent. The monomers that are used to synthesize polymers of thisembodiment are suitable for use as the monomers fed to a DNAsynthesizer, as described below. Thus, the polymer can be synthesizedvery specifically. Further, the additional incorporation ofpolynucleotide sequences, polyether blocks, and lipophilic substituentscan be done using the advanced automation developed for polynucleotidesyntheses. The fifth embodiment also encompasses this method ofsynthesizing a polycationic polymer.

In yet another embodiment, the invention relates to a polymer of aplurality of covalently bound polymer segments wherein said segmentscomprise (a) at least one polycation segment which segment is a cationichomopolymer, copolymer, or block copolymer comprising at least threeaminoalkylene monomers, said monomers being selected from the groupconsisting of:

(i) at least one tertiary amino monomer of the formula:

and the quaternary salts of said tertiary amino monomer, and (ii) atleast one secondary amino monomer of the formula:

and the acid addition and quaternary salts of said secondary aminomonomer, in which:

R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a Bmonomer; each of R² and R³, taken independently of the other, is thesame or different straight or branched chain alkanediyl group of theformula:

—(C_(z)H_(2z))—

in which z has a value of from 2 to 8; R⁴ is hydrogen satisfying onebond of the depicted geminally bonded carbon atom; and R⁵ is hydrogen,alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁶ ishydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁷is a straight or branched chain alkanediyl group of the formula:

—(C_(z)H_(2z))—

in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkyl of 2 to8 carbon atoms, an A monomer, or a B monomer; and

(b) at least one straight or branched chained polyether segment havingfrom about 5 to about 400 monomeric units which is:

(i) a homopolymer of a first alkyleneoxy monomer —OC_(n)H_(2n)— or

(ii) a copolymer or block copolymer of said first alkyleneoxy monomerand a second different alkyleneoxy monomer —OC_(m)H_(2m)—, in which nhas a value of 2 or 3 and m has a value of from2to 4.

The preferred polycationic segments include but are not limited topolyamines (e.g., spermine, polyspermine, polyethyleneimine,polypropyleneimine, polybutileneimine, polypentyleneimine,polyhexyleneimine and copolymers thereof), copolymers of tertiary aminesand secondary amines, partially or completely quaternized amines,polyvinyl pyridine and the quaternary ammonium salts of these polycationsegments. Preferred polycation segments also include aliphatic,heterocyclic or aromatic ionenes (Rembaum et al. Polymer letters, 1968,6; 159; Tsutsui, T., In Development in ionic polymers -2, Wilson A. D.and Prosser, H. J. (eds.) Applied Science Publishers, London, New York,vol. 2, pp. 167-187, 1986). Particularly preferred polycationic segmentsinclude a plurality of cationic repeating units of formula —N—R⁰,wherein R⁰ is a straight chain aliphatic group of 2 to 6 carbon atoms,which may be substituted. Each —N—R⁰— repeating unit in an polycationsegment can be the same or different from another —N—R⁰— repeating unitin the segment.

The polycationic segments in the copolymers of the invention can bebranched. For example, polyspermine-based copolymers are branched. Thecationic segment of these copolymers was synthesized by condensation of1,4-dibromobutane and N-(3-aminopropyl)-1,3-propanediamine. Thisreaction yields highly branched polymer products with primary,secondary, and tertiary amines.

An example of branched polycations are products of the condensationreactions between polyamines containing at least 2 nitrogen atoms andalkyl halides containing at least 2 halide atoms (including bromide orchloride). In particular, the branched polycations are produced as aresult of polycondensation. An example of this reaction is the reactionbetween N-(3-aminiopropyl)-1,3-propanediamine and 1,4-dibromobutane,producing polyspermine.

Another example of a branched polycation is polyethyleneiminerepresented by the formula:

(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

Additionally, cationic dendrimers, for example, polyamidoamines orpolypropyleneimines of various generations (i.e., molecular weight),(Tomalia et al., Angew. Chem., Int. Ed. Engl. 1990, 29, 138) can be alsoused as polycation segments of block copolymers for gene delivery.

In yet another embodiment, the invention relates to a polymer of aplurality of covalently bound polymer segments wherein the segmentscomprise:

(a) at least one polycation segment which is a cationic homopolymer orcopolymer comprising at least three cationic amino acids, or at leastthree aminoalkylene monomers, the monomers selected from the groupconsisting of:

(i) at least one tertiary amino monomer of the formula:

and the quaternary salts of said tertiary amino monomer, and (ii) atleast one secondary amino monomer of the formula:

and the acid addition and quaternary salts of said secondary aminomonomer, in which:

R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a Bmonomer; each of R² and R³, taken independently of the other, is thesame or different straight or branched chain alkanediyl group of theformula:

—(C_(z)H_(2z))—

in which z has a value of from 2 to 8; R⁴ is hydrogen satisfying onebond of the depicted geminally bonded carbon atom; and R⁵ is hydrogen,alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁶ ishydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁷is a straight or branched chain alkanediyl group of the formula:

—(C_(z)H_(2z))—

in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkyl of 2 to8 carbon atoms, an A monomer, or a B monomer; and

(b) at least one water-soluble nonionic polymer segment. This includesat least one nonionic polymer segment comprising at least three of thesame or different repeating units containing at least one atom selectedfrom the group consisting of oxygen and nitrogen.

In this embodiment the polycation serves as the binding segment, whilethe nonionic polymer serves as a lypohilizing segment.

The polycation segments preferred in this embodiment are the same aspolycations preferred in the previous embodiments. These preferredpolycation segments include but are not limited to polyamines (e.g.,spermine, polyspermine, polyethyleneimine, polypropyleneimine,polybutilene-imine, poolypentyleneimine, polyhexyleneimine andcopolymers thereof), copolymers of tertiary amines and secondary amines,partially or completely quaternized amines, polyvinyl pyridine and thequaternary ammonium salts of said polycation segments.

It is preferred that nonionic polymer segments comprise water-solublepolymers, which are nontoxic and nonimunogenic. The preferred nonionicpolymer segment is at least one water-soluble nonionic polymer segmentis a homopolymer or copolymer of at least one of the monomers selectedfrom the group consisting of acrylamide, gycerol, vinylalcohol,vinylpyrrolidone, vinylpyridine, vinylpyridine N-oxide, oxazoline, or aacrifoge morpholine, and derivatives thereof. This includes for examplepolyacrylamides, polygycerols, polyvinylalcohols, polyvinylpyrrolidones,polyvinylpyridine N-oxides, copolymers of vinylpyridine N-oxide andvinylpyridine, polyoxazolines, polyacroylmorpholines or derivativesthereof. Nonionic segments comprising products of polymerization ofvinyl monomers are also preferred, including but not limiting to thefollowing nonionic polymer segments and derivatives thereof:

in which m a value of from 3 to about 10,000.

Included within the scope of the invention are compositions comprisingthese polymers and a suitable targeting molecule. Also included withinthe scope of the invention are compositions comprising polymer, apolynuclcotide, and a surfactant. The invention also relates tocopolymers comprising at least one polynucleotide segment and at leastone polyether segment, said polyether segment comprising oxyethylene andoxypropylene.

The present compositions can be used in a variety of treatments. Forexample, the compositions can be used in Gene therapy including genereplacement or excision therapy, and gene addition therapy, (B. Huber,Gene therapy for neoplastic diseases; B E Huber and J S Lazo Eds., TheNew York Academy of Sciences, NY, N.Y., 1994, pp. 6-11). Also, antisensetherapy targets genes in the nucleus and/or cytoplasm of the cell,resulting in their inhibition (Stein and Cheng, Science 261:1004, 1993;De Mesmaeker et al. Acc. Chem. Res. 28:366, 1995). Aptamer nucleic aciddrugs target both intra- and extracellular proteins, peptides and smallmolecules. See Ellington and Szostak, Nature (London), 346,818, 1990.Antigen nucleic acid compounds can be used to target duplex DNA in thenucleus. See Helene and Tolume, Biochim, Biophys. Acta 1049:99, 1990.Catalytic polynucleotides target mRNA in the nucleus and/or cytoplasm(Cech, Curr. Opp. Struct. Biol. 2:605, 1992).

Examples of genes to be replaced, inhibited and/or added include, butare not limited to, adenosine deaminase, tumor necrosis factor, cellgrowth factors, Factor IX, interferons (such as α-, β- and γ-interferon), interleukins (such interleukin 2,4, 6 and 12), HLA-B7,HSV-TK, CFTR, HIV -1, β-2, microglobulin, retroviral genes (such as gag,pol, env, tax, and rex), cytomegalovirus, herpes viral genes (such asherpes simplex virus type I and II genes ICP27/UL54, ICP22/US1,ICP/IE175, protein kinase and exonuclease/UL13, protein kinase/US3,ribonuclease reductase ICP6/UL39, immediate early (IE) mRNAIE3/IE175/ICP4, 1E4/ICP22/US1, IE5/ICP47, IE110, DNA polymerase/UL30,UL13), human multidrug resistance genes (such as mdrl), oncogenes (suchas H-c-ras, c-myb, c-myb, bcl-2, bcr/abl), tumor suppressor gene p53,human MHC genes (such as class 1 MHC), immunoglobulins (such as IgG,IgM, IgE, IgA), hemoglobin α- and β- chains, enzymes (such as carbonicanhydrase, triosephoshate isomerase, GTP-cyclhydrdolase I, phenylalaninehydrolase, sarcosine dehydrogenase, glucocerobrosidase,glucose-6-phosphate dehydrogenase), dysotrophin, fibronectin,apoliprotein E, cystic fibrosis transmembrane conductance protein, c-srcprotein, V(D)J recombination activating protein, immunogenes, peptideand protein antigens (“DNA vaccine”) and the like.

Genetic diseases can also be treated by the instant compositions. Suchdiseases include, but are not limited to, rheumatoid arthritis,psoriasis, Crohn's disease, ulcerative colitis, α-thalassemia,β-thalassemia, carbonic anhydrase II deficiency syndrome,triosephosphate isomerase deficiency syndrome,tetrahydrobiopterindeficient hyperphenylalaniemia, classicalphenylketonuria, muscular dystrophy such as Duchenne Muscular Dystrophy,hypersarkosinemia, adenomatous intestinal polyposis, adenosine deaminasedeficiency, malignant melanoma, glucose-6-phosphste dehydrogenasedeficiency syndrome, arteriosclerosis and hypercholesterolemia,Gaucher's disease, cystic fibrosis, osteopetrosis, increased spontaneoustumors, T and B cell immunodeficiency, high cholesterol, arthritisincluding chronic rheumatoid arthritis, glaucoma, alcoholism and thelike.

The compositions can also be used to treat neoplastic diseasesincluding, but not limited to, breast cancer (e.g., breast, pancreatic,gastric, prostate, colorectal, lung, ovarian), lymphomas (such asHodgkin and non-Hodgkin lymphoma), melanoma and malignant melanoma,advanced cancer hemophilia B, renal cell carcinoma, gliblastoma,astrocytoma, gliomas, AML and CML and the like.

Additionally, the compositions can be used to treat (i) cardiovasculardiseases including but not limited to stroke, cardiomyopathy associatedwith Duchenne Muscular Dystrophy, myocardial ischemia, restenosis andthe like, (ii) infectious diseases such as Hepatitis, HIV infections andAIDS, Herpes, CMV and associated diseases such as CMV renitis, (iii)transplantation related disorders such as renal transplant rejection andthe like, and (iv) are useful in vaccine therapies and immunization,including but not limited to melanoma vaccines, HIV vaccines, malaria,tuberculosis, and the like.

Target Cells

Cell targets can be ex vivo and/or in vivo, and include T and Blymphocytes, primary CML, tumor infiltrating lymphocytes, tumor cells,leukemic cells (such as HL-60, ML-3, KG-1 and the like), skinfibroblasts, myoblasts, cells of central nervous system includingprimary neurons, liver cells, carcinoma (such as Bladder carcinoma T24,human colorectal carcinoma Caco-2), melanoma, CD34+ lymphocytes, NKcells, macrophages, hemotopoetic cells, neuroblastoma (such as LAN-5 andthe like), gliomas, lymphomas (such as Burkitt lymphomas ST486), JD38),T-cell hybridomas, muscle cells such as primary smooth muscle, and thelike.

Filed concurrently with the parent of this application (Nov. 18, 1994)was Ser. No. 08/342,079 entitled “POLYMER LINKED BIOLOGICAL AGENTS”. Theentire disclosure of that application is incorporated herein byreference.

The degree of polymerization of the hydrophilic (A-type) segments or thehydrophobic (B-type) segments of formulas (I)-(XIII) can preferably bebetween about 5 and about 400. More preferably, the degree ofpolymerization is between about 5 and about 200, still more preferably,between about 5 and about 80. The degree of polymerization of the R-typepolycation segments can preferably be between about 2 and about 300.More preferably, the degree of polymerization is between about 5 andabout 180, still more preferably, between about 5 and about 60. Thedegree of polymerization of the polycationic polymer can preferably bebetween about 10 and about 10,000. More preferably, the degree ofpolymerization is between about 10 and about 1,000, still morepreferably, between about 10 and about 100.

The repeating units that comprise the segments, for A-type, B-type andR-type segments, will generally have molecular weight between about 30and about 500, preferably between about 30 and about 100, still morepreferably between about 30 and about 60. Generally, in each of theA-type or B-type segments, at least about 80% of the linkages betweenrepeating units are ether linkages, preferably, at least about 90% areether linkages, more preferably, at least about 95% are ether linkages.Ether linkages, for the purposes of this application, encompassglycosidic linkages (i.e., sugar linkages). However, in one aspect,simple ether linkages are preferred.

The polynucleotide component (pN) of formulas (IX) through (XIII) willpreferably comprise from about 5 to about 1,000,000 bases, morepreferably about 5 to about 100,000 bases, yet more preferably about 10to about 10,000 bases.

The polycation segments have several positively ionizable groups and anet positive charge at physiologic pH. The polyether/polycation polymersof formulas (V)-(VIII) can also serve as polycationic polymers.Preferably, the polycation segments have at least about 3 positivecharges at physiologic pH, more preferably, at least about 6, still morepreferably, at least about 12. Also preferred are polymers or segmentsthat, at physiologic pH, can present positive charges with a distancebetween the charges of about 2 Å to about 10 Å. The distancesestablished by ethtyleneimine, aminopropylene, aminobutilene,aminopentylene and aminohehhylene repeating units, or by mixtures of atleast two of these groups are most preferred. Preferred are polycationicsegments that utilize (NCH₂CH₂), (NCH₂CH₂CH₂), (NCH₂CH₂CH₂CH₂),(NCH₂CH₂CH₂CH₂CH₂), and (NCH₂CH₂CH₂CH₂CH₂CH₂) repeating units, or amixture thereof.

Polycation segments having an —N—R⁰— repeating unit are also preferred.R⁰ is preferably an ethylene, propylene, butylene, pentylene, orhexylene which can be modified. In a preferred embodiment, in at leastone of the repeating units R⁰ includes a DNA intercalating group such asan ethidium bromide group. Such intercalating groups can increase theaffinity of the polymer for nucleic acid. Preferred substitutions on R⁰include alkyl of 1-6 carbon atoms, hydroxy, hydroxyalkyl, wherein thealkyl has 1-6 carbon atoms, alkoxy having 1-6 carbon atoms, an alkylcarbonyl group having 2-7 carbon atoms, alkoxycarbonyl wherein thealkoxy has 1-6 carbon atoms, alkoxycarbonylalkyl wherein the alkoxy andalkyl each independently has 1-6 carbon atoms, alkylcarboxyalkyl whereineach alkyl group has 1-6 carbon atoms, aminoalkyl wherein the alkylgroup has 1-6 carbon atoms, alkylamino or dialkylamino where each alkylgroup independently has 1-6 carbon atoms, mono- or di-alkylaminoalkylwherein each alkyl independently has 1-6 carbon atoms, chloro,chloroalkyl wherein the alkyl has from 1-6 carbon atoms, fluoro,fluoroalkyl wherein the alkyl has from 1-6 carbon atoms, cyano, or cyanoalkyl wherein the alkyl has from 1-6 carbon atoms or a carboxyl group.More preferably, R⁰ is ethylene, propylene or butylene.

Polymers according to the first embodiment of the invention areexemplified by the block copolymers having the formulas:

in which x, y, z, i and j have values from about 5 to about 400,preferably from about 5 to about 200, more preferably from about 5 toabout 80, and wherein for each R¹, R² pair, one is hydrogen and theother is a methyl group.

Formulas (XIV) through (XVI) are oversimplified in that, in practice,the orientation of the isopropylene radicals within the B segment willbe random. This random orientation is indicated in formula (XVII), whichis more complete. Such poly(oxyethylene)-poly(oxypropylene) compoundshave been described by Santon, Am. Perfumer Cosmet., 72(4):54-58 (1958);Schmolka, Loc. cit. 82(7):25 (1967); Schick, Non-ionic Surfactants, pp.300-371 (Dekker, NY, 1967). A number of such compounds are commerciallyavailable under such generic trade names as “poloxamers”, “pluronics”and “synperonics.” Pluronic polymers within the B-A-B formula are oftenreferred to as “reversed” pluronics, “pluronic R” or “meroxapol”. The“polyoxamine” polymer of formula (XVII) is available from BASF(Wyandotte, Mich.) under the tradename Tetronic™. The order of thepolyoxyethylene and polyoxypropylene segments represented in formula(XVII) can be reversed, creating Tetronic R™, also available from BASF.See, Schmolka, J. Am. Oil Soc., 59:110 (1979).Polyoxypropylene-polyoxyethylene block copolymers can also be designedwith hydrophilic segments comprising a random mix of ethylene oxide andpropylene oxide repeating units. To maintain the hydrophilic characterof the segment, ethylene oxide will predominate. Similarly, thehydrophobic segment can be a mixture of ethylene oxide and propyleneoxide repeating units. Such block copolymers are available from BASFunder the tradename Pluradot™.

The diamine-linked pluronic of formula (XVII) can also be a member ofthe family of diamine-linked polyoxyethylene-polyoxypropylene polymersof formula:

wherein the dashed lines represent symmetrical copies of the polyetherextending off the second nitrogen, R* is an alkylene of 2 to 6 carbons,a cycloalkylene of 5 to 8 carbons or phenylene, for R¹ and R²,either,(a) both are hydrogen or (b) one is hydrogen and the other ismethyl, for R³ and R⁴ either (a) both are hydrogen or (b) one ishydrogen and the other is methyl, if both of R³ and R⁴ are hydrogen,then one R⁵ and R⁶ is hydrogen and the other is methyl, and if one of R³and R⁴ is methyl, then both of R⁵ and R⁶ are hydrogen.

Those of ordinary skill in the art will recognize, in light of thediscussion herein, that even when the practice of the invention isconfined for example, to poly(oxyethylene)-poly(oxypropylene) compounds,the above exemplary formulas are too confining. Thus, the units makingup the first segment need not consist solely of ethylene oxide.Similarly, not all of the B-type segment need not consist solely ofpropylene oxide units. Instead, the segments can incorporate monomersother than those defined in formulas (XIV)-(XVII), so long as theparameters of the first embodiment are maintained. Thus, in the simplestof examples, at least one of the monomers in segment A might besubstituted with a side chain group as previously described.

In another aspect, the invention relates to a polynucleotide complexcomprising a block copolymer at least one of formulas (I)-(XIII),wherein the A-type and B-type segments are substantially made up ofrepeating units of formula —O—R⁹, where R⁹ is:

(1) —(CH₂)_(n)—CH(R⁶), wherein n is an integer from 0 to about 5 and R⁶is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having 1-6 carbonatoms, phenyl, alkylphenyl wherein the alkyl has 1-6 carbon atoms,hydroxy, hydroxyalkyl, wherein the alkyl has 1-6 carbon atoms, alkoxyhaving 1-6 carbon atoms, an alkyl carbonyl group having 2-7 carbonatoms, alkoxycarbonyl, wherein the alkloxy has 1-6 carbon atoms,alkoxycarbonylalkyl, wherein the alkoxy and alkyl each independently has1-6 carbon atoms, alkylcarboxyalkyl, wherein each alkyl group has 1-6carbon atoms, aminoalkyl wherein the alkyl group has 1-6 carbon atoms,alkylamine or dialkylamino, wherein each alkyl independently has 1-6carbon atoms, mono- or di-alkylaminoalkyl wherein each alkylindependently has 1-6 carbon atoms, chloro, chloroalkyl wherein thealkyl has from 1-6 carbon atoms, fluoro, fluoroalkyl wherein the alkylhas from 1-6 carbon atoms, cyano or cyano alkyl wherein the alkyl hasfrom 1-6 carbon atoms or carboxyl; (2) a carbocyclic group having 3-8:ring carbon atoms, wherein the group can be for example, cycloalkyl oraromatic groups, and which can include alkyl having 1-6 carbon atoms,alkoxy. having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms,dialkylamino wherein each alkyl independently has 1-6 carbon atoms,amino, sulfonyl, hydroxy, carboxy, fluoro or chloro substitutions, or(3) a heterocyclic group, having 3-8 ring atoms, which can includeheterocycloalkyl or heteroaromatic groups, which can include from 1-4heteroatoms selected from the group consisting of oxygen, nitrogen,sulfur and mixtures thereto, and which can include alkyl having 1-6carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6carbon atoms, dialkylamino wherein each alkyl independently has 1-6carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro or chlorosubstitutions.

Preferably, n is an integer from 1 to 3. The carbocyclic or heterocyclicgroups comprising R⁵ preferably have from 4-7 ring atoms, morepreferably 5-6. Heterocycles preferably include from 1-2 heteroatoms,more preferably, the heterocycles have one heteroatom. Preferably, theheterocycle is a carbohydrate or carbohydrate analog. Those of ordinaryskill will recognize that the monomers required to make these polymersare synthetically available. In some cases, polymerization of themonomers will require the use of suitable protective groups, as will berecognized by those of ordinary skill in the art. Generally, the A- andB-type segments are at least about 80% comprised of —OR⁵— repeatingunits, more preferably at least about 90%, yet more preferably at leastabout 95%.

In another aspect, the invention relates to a polynucleotide complexcomprising a block copolymer of one of formulas (I)-(XIII) wherein theA-type and B-type segments consist essentially of repeating units offormula —O—R⁵ wherein R⁷ is a C to C alkyl group.

A wide variety of nucleic acid molecules can be the nucleic acidcomponent of the composition. These include natural and synthetic DNA orRNA molecules and nucleic acid molecules that have been covaleritlymodified (to incorporate groups including lipophilic groups,photo-induced crosslinking groups, alkylating groups, organometallicgroups, intercalating groups, lipophilic groups, biotin, fluorescent andradioactive groups, and groups that modify the phosphate backbone). Suchnucleic acid molecules can be, among other things, antisense nucleicacid molecules, gene-encoding DNA (usually including an appropriatepromoter sequence), ribozymes, aptamers, antigen nucleic acids,oligonucleotide α-anomers, ethylphosphotriester analogs,alkylphosphomates, phosphorothionate and phosphorodithionateoligonucleotides, and the like. In fact, the nucleic acid component canbe any nucleic acid that can beneficially be transported into a cellwith greater efficiency, or stabilized from degradative processes, orimproved in its biodistribution after administration to an animal.

Examples of useful polymers pursuant to formulas (V)-(VIII) include thepoly(oxyethylene)-poly-L-lysine) diblock copolymer of the followingformula:

wherein i is an integer of from about 5 to about 100, and j is aninteger from about 4 to about 100. A second example is thepoly(oxyethylene)-poly-(L-alanine-L-lysine) diblock copolymer offormula:

wherein i is an integer of from about 5 to about 100, and j is aninteger from about 4 about 100. A third example is thepoly(oxyethylene)-poly(propyleneimine/butyleneimine) diblock copolymerof the following formula:

wherein i is an integer from about 5 about 200 and j is an integer from1 to about 10. A fourth example is thepoly(oxyethylene)-poly(N-ethyl-4-vinylpyridinium bromide)(“pOE-pEVP-Br”) of formula:

wherein i is an integer of from about 5 to about 100 and j is an integerof from about 10 to about 500. Still another example is the polymer offormula:

CH₃O—(CH₂CH₂O)_(i)CO[(NH(CH₂)₃)₂NH(CH₂)₄]_(j)—(NH(CH₂)₃)₂—NCHO—O—(CH₂CH₂O)_(k)—CH₃  (XXII)

wherein i is an integer from about 10 to about 200, j is an integer fromabout 1 to about 8, and k is an integer from about 10 to about 200.Still another example is the polymer of formula:

H—G_(j)—(NH(CH₂)₃)₂—N—NH—CO—O—(CH₂CH₂O)_(i)CO—G_(m)—(NH(CH₂)₃)₂—NH₂  (XXIII)

wherein “G” comprises —(NH(CH₂)₃)₃—CH₂NH₂—, i and j are as defined forformula (XVIII), and m is an integer from about 1 to about 8.

The block copolymers utilized in the invention will typically, undercertain circumstances, form micelles or micelle-like aggregates of fromabout 10 nm to about 100 nm in diameter. Micelles are supramolecularcomplexes of certain amphiphilic molecules that form in aqueoussolutions due to microphase separation of the nonpolar portions of theamphiphiles. Micelles form when the concentration of the amphiphilereaches, for a given temperature, a critical micellar concentration(“CMC”) that is characteristic of the amphiphile. Such micelles willgenerally include from about 10 to about 300 block copolymers. Byvarying the sizes of the hydrophilic and hydrophobic portions of theblock copolymers, the tendency of the copolymers to form micelles atphysiological conditions can be varied. The micelles have a dense coreformed by the water insoluble repeating units of the B blocks andcharge-neutralized nucleic acids, and a hydrophilic shell formed by theA segments. The micelles have translational and rotational freedom insolution, and solutions containing the micelles have low viscositysimilar to water. Micelle formation typically occurs at copolymerconcentrations from about 0.001 to 5% (w/v). Generally, theconcentration of polycationic polymers and polynucleic acid will be lessthan the concentration of copolymers in the polynucleotide compositions,preferably at least about 10-fold less, more preferably at least about50-fold.

At high concentrations, some of the block copolymers utilized in theinvention will form gels. These gels are viscous systems in which thetranslational and rotational freedom of the copolymer molecules issignificantly constrained by a continuous network of interactions amongcopolymer molecules. In gels, microsegregation of the B segmentrepeating units may or may not occur. To avoid the formation of gels,polymer concentrations (for both block copolymers andpolyether/polycation polymers) will preferably be below about 15% (w/v),more preferably below about 10%, still more preferably below about 5%.In the first embodiment of the invention, it is more preferred that gelsbe avoided.

When the polynucleotide composition includes cationic components, thecations will associate with the phosphate groups of the polynucleotide,neutralizing the charge on the phosphate groups and rendering thepolynucleotide component more hydrophobic. The neutralization ispreferably supplied by cations on R-type polymeric segments or onpolycationic polymers. However, the phosphate charge can also beneutralized by chemical modification or by association with ahydrophobic cations such as N-[1-(2,3-dioleyloxy)-N,N′-3-methylammoniurnchloride]. In aqueous solution, the charge neutralized polynucleotidesare believed to participate in the formation of supramolecular,micelle-like particles, termed “polynucleotide complexes.” Thehydrophobic core or the complex comprises the charge neutralizedpolynucleotides and the B-type segments. The hydrophilic shell comprisesthe A-type segments. The size of the complex will generally vary fromabout 10 nm to about 100 nm in diameter. In some contexts it ispractical to isolate the complex from unincorporated components. Thiscan be done for instance, by gel filtration chromatography.

The ratio of the components of the polynucleotide composition is animportant factor in optimizing the effective transmembrane permeabilityof the polynucleotides in the composition. This ratio can be identifiedas ratio Ø, which is the ratio of positively charged groups tonegatively charged groups in the composition at physiological pH. IfØ<1, the complex contains non-neutralized phosphate from thepolynucleotide. The portions of the polynucleotides adjacent to thenon-neutralized charges are believed to be a part of the shell of apolynucleotide complex. Correspondingly, if Ø>1, the polycationicpolymer or R-type segment will have non-neutralized charges, and theun-neutralized portions will fold so that they form a part of the shellof the complex. Generally, Ø will vary from about 0 (where there are nocationic groups) to about 100, preferably Ø will range between about0.01 and about 50, more preferably, between about 0.1 and about 20. Øcan be varied to increase the efficiency of transmembrane transport and,when the composition comprises polynucleotide complexes, to increase thestability of the complex. Variations in Ø can also affect thebiodistribution of the complex after administration to an animal. Theoptimal Ø will depend on, among other things, (1) the context in whichthe polynucleotide composition is being used, (2) the specific polymersand oligonucleotides being used, (3) the cells or tissues targeted, and(4) the mode of administration.

It will in some circumstances be desirable to incorporate in thepolynucleotide compositions of the current invention, by noncovalentassociation or covalent conjugation, targeting molecules. See forexample, Kabanov et al, J. Controlled Release, 22:141 (1992), thecontents of which are hereby incorporated by reference. The term“targeting molecule” means any molecule, atom, or ion that enhancesbinding, transport, accumulation, residence time, bioavailability ormodifies biological activity of the polynucleotides or thepolynucleotide compositions of the current invention in the body orcell. The targeting molecule will frequently comprise an antibody,fragment of antibody or chimeric antibody molecule typically withspecificity for a certain cell surface antigen. The targeting moleculecan also be, for instance, a hormone having a specific interaction witha cell surface receptor, or a drug having a cell surface receptor. Forexample, glycolipids can serve to targetia polysaccharide receptor. Thetargeting molecules can also be, for instance, enzymes, lectins, andpolysaccharides low molecular mass ligands, such as folic acid andderivatives thereof are also useful in the context of the currentinvention. The targeting molecules can also be polynucleotide,polypeptide, peptidomimetic, carbohydrates including polysaccharides,derivatives thereof or other chemical entities obtained by means ofcombinatorial chemistry and biology. The targeting molecules can be usedto facilitate intracellular transport of the polynucleotide compositionsof the invention, for instance transport to the nucleus, by using, forexample, fusogenic peptides as targeting molecules described bySoukchareun et al., Bioconjugate Chem., 6, 43 (1995), or Arar et al.,Bioconjugate Chem., 6, 43 (1995), caryotypic peptides, or otherbiospecific groups providing site-directed transport into a cell (inparticular, exit from endosomic compartments into cytoplasm, or deliveryto the nucleus).

Included within the scope of the invention are compositions comprisingthe polynucleotide, block copolymer of the current invention and asuitable targeting molecule. The targeting molecule can be covalentlylinked to any of the polymer segments of the block copolymers identifiedherein (or polynucleotide complexes thereof), including cationic andnonionic polymer segments. For instance, the targeting molecule can belinked to the free-terminal or pendant groups of the nonionic segments.Such targeting molecules can be linked to the terminal or pendant —OHend group of the polymer segments, and the terminal or pendant —NH₂group of the polymer segments, or the terminal or pendant —COOH endgroup of the polymer segments, or the like.

It will in some circumstances be desirable to incorporate targetingmolecules through ligand-receptor constructs, in which:

(i) the ligand molecule is a chemical entity (e.g., a molecule, atom, orion) capable of specific binding with the receptor molecule; (ii) thereceptor molecule is a chemical entity capable of specific binding tothe ligand molecule; or (iii) the ligand molecules, receptor molecules(or both) are incorporated into the block copolymers (or polynucleotidecomplexes thereof), targeting molecules, or both. This is done bynoncovalent association or covalent conjugation so that after (i) mixingtargeting molecules and block copolymers (or polynucleotide complexesthereof) with the ligand and receptor molecules attached to them, or(ii) by adding either free ligand or receptor (or both) to the mixtureof targeting molecules and block copolymers (or polynucleotide complexesthereof), the targeting molecule becomes attached to the blockcopolymers (or to the polynucleotide complexes thereof) throughligand-receptor binding. Examples of such constructs include constructsusing biotin as the ligand and avidin or streptavidin as the receptor.For example, biotin or a derivative thereof can be covalently linked tothe block copolymers (or polynucleotide complexes thereof) of thecurrent invention and avidin (or streptavidin) can be covalently linkedto the targeting molecule. Alternatively, biotin can be linked to boththe block copolymers (or polynucleotide complexes thereof) and targetingmolecule, and the latter can be linked through avidin, which has fourbiotin-binding centers. Further, additional complex constructscomprising biotin and avidin can be used for incorporating targetingmolecules in polynucleotide compositions of the current invention. Inone particular embodiment, the invention provides for the blockcopolymers (or polynucleotide complexes thereof) with biotin moleculesor derivatives thereof linked to at least one polycation or nonionicpolymer segments or both polycation and nonionic polymer segments. Thoseof ordinary skill in the art will ecognize, in light of the discussionherein, that even when the practice of the invention is confined forexample, to biotin-avidin or biotin-streptavidin constructs or thesimilar constructs, there are numerous ways available providing for thedesign of the ligand-receptor constructs with the desiredcharacteristics pursuant to this invention. Such constructs, forexample, can comprise ligands and/or receptors that are polynucleotide,polypeptide, peptidomimetic, carbohydrates including polysaccharides,derivatives thereof or other chemical entities obtained by means ofcombinatorial chemistry and biology.

The targeting molecules which can be associated with the polynucleotidecompositions of the invention can also have a targeting group havingaffinity for a cellular site and a hydrophobic group. Such targetingmolecules can provide for the site specific delivery and recognition inthe body. The targeting molecule spontaneously associates with thepolynucleotide complex and be “anchored” thereto through the hydrophobicgroup. These targeting adducts will typically comprise about 1% or lessof the polymers in a final composition. In the targeting molecule, thehydrophobic group can be, among other things, a lipid group such as afatty acyl group. Alternatively, it can be an ionic or nonionichomopolymer, copolymer, block copolymer, graft copolymer, dendrimer oranother natural or synthetic polymer.

In the targeting molecule, the hydrophobic group can be, among otherthings, a lipid group such as a fatty acyl group. Alternately, it can bea block copolymer or another natural synthetic polymer. The targetinggroup of the targeting molecule will frequently comprise an antibody,typically with specificity for a certain cell surface antigen. It couldalso be, for instance, a hormone having a specific interaction with acell surface receptor, or a drug having a cell surface receptor. Forexample, glycolipids could serve to target a polysaccharide receptor. Itshould be noted that the targeting molecule can be attached to any ofthe polymer segments identified herein, including R-type polymericsegments and to the polycationic polymers. For instance, the targetingmolecule can be covalently attached to the free-terminal groups of thepolyether segment of the block copolymer of the invention. Suchtargeting molecules can be covalently attached to the —OH end group ofthe polymers of the formulas XVIII, XIX, XX, and XXI, and the —NH₂ endgroup of the polymers of formulas XVIII (preferably the ε-amino group ofthe terminal lysyl residue), XX or XXIII, or the —COOH end group of thepolymers of formulas XVIII and XIX. Targeting molecules can be used tofacilitate intracellular transport of the polynucleotide composition,for instance transport to the nucleus, by using, for example, fusogenicpeptides as targeting molecules described by Soukchareun et al.,Bioconjugate Chem., 6, 43, 1995 or Arar et al., Bioconjugate Chem., 6,43, 1995, caryotypic peptides, or other biospecific groups providingsite-directed transport into a cell (in particular, exit from endosomiccompartments into cytoplasm, or delivery to the nucleus).

The polynucleotide component of the compositions of the invention can beany polynucleotide, but preferably a polynucleotide with at least about3 bases, more preferably at least about 5 bases. Still more preferredare at least 10 bases. Included among the suitable polynucleotides areviral genoimes and viruses (including the lipid or protein viral coat).This includes viral vectors including, but not limited to, retroviruses,adenoviruses, herpes-virus, and Pox-viruses. Other suitable viralvectors for use with the present invention will be obvious to thoseskilled in the art. The terms “poly(nucleic acid)” and “polynucleotide”are used interchangeably herein. An oligonucleotide is a polynucleotide,as are DNA and RNA.

A polynucleotide derivative is a polynucleotide having one or moremoieties (i) wherein the moieties are cleaved, inactivated or otherwisetransformed so that the resulting material can function as apolynucleotide, or (ii) wherein the moiety does not prevent thederivative from functioning as a polynucleotide.

For polyethylene oxide-polypropylene oxide copolymers, thehydrophilic/hydrophobic properties, and micelle forming properties of ablock copolymer are, to a certain degree, related to the value of theratio, n. The ratio, n, is defined as:

 n=(|B|/|A|)x(b/a)=(|B|/|A|)x1.32

where |B| and |A| are the number of repeating units, in the hydrophobicand hydrophilic segments of the copolymer, respectively, and b and a arethe molecular weights for the respective repeating units. The value of nwill typically be between about 0.2 and about 9.0, more preferably,between about 0.2 and about 1.5. Where mixtures of block copolymers areused, n will be the weighted average of n for each contributingcopolymers, with the averaging based on the weight portions of thecomlponent copolymers. When copolymers other than polyethyleneoxide-polypropylene oxide copolymers are used, similar approaches can bedeveloped to relate the hydrophobic/hydrophilic properties of one memberof the class of polymers to the properties of another member of theclass.

Surfactant-Containing Polynucleotide Compositions

The invention also includes compositions of polynucleotide, cationiccopolymer, and a suitable surfactant. The surfactant, should be (i)cationic (including those used in various transfection cocktails), (ii)non ionic (e.g., Pluronic or Tetronic), or (iii) zwitterionic (includingbetains and phospholipids). These surfactants increase solubility of thecomplex and increase biological activity of the compositions.

Cationic surfactants include but are not limited to primary amines,secondary amines, tertiary amines (e.g.,N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane), quaternaryamine salts (e.g., dodecyltrimethylammonium bromide,hexadecyltrimethylammonium bromide, mixed alkyl-trimethylammoniumbromide, tetradecyltrimethylammonium bromide, benzalkonium chloride,benzethonium chloride, benzyldimethyldodecylammonium chloride,benzyl-dimethylhexadecylammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide, methylbenzethonium chloride, decamethonium chloride,methyl mixed trialkyl ammonium chloride, methyl trioctylamimoniumchloride),N,N-dimethyl-N-[2-(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy)ethyl]-benzenemethanaminiumchloride (DEBDA), dialkyldimetylammonium salts,N-[1-(2,3-dioleyloxy)-propyl]-N,N,N,-trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio)propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl),1,2-diacyl-3-(dimethylammonio)propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester,cholesteryl (4′-trimethylammonio) butanoate), N-alkyl pyridinium salts(e.g. cetylpyridinium bromide and cetylpyridinium chloride),N-alkylpiperidinium salts, dicationic bolaform electrolytes (C₁₂Me₆;C₁₂Bu₆), dialkylglycetyl-phosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine), cholesterol hemisuccinate choline ester,lipopolyamines (e.g., dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES), lipopoly-L(or D)-lysine (LPLL,LPDL), poly(L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives of cholesterol(e.g., cholesteryl-3β-oxy-succinamidoethylenetrimethylammonium salt,cholesteryl-3β-oxysuccinamidoethylenedimethyl-amine,cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt,cholesteryl-3β-carboxy-amidoethylenedimethylamine,3β[N-(N′,N′-dimethylaminoetane-carbomoil]cholesterol).

Non-ionic surfactants include but are not limited to n-Alkylphenylpolyoxyethylene ether, n-alkyl polyoxyethylene ethers (e.g., Tritons™),sorbitan esters (e.g., Spans™), polyglycol ether surfactants(Tergitol™), polyoxyethylenesorbitan (e.g., Tweens™), polysorbates,poly-oxyethylated glycol monoethers (e.g., Brij™, polyoxylethylene 9lauryl ether, polyoxylethylene 10 ether, polyoxylethylene 10 tridecylether), lubrol, copolymers of ethylene oxide and propylene oxide (e.g.,Pluronic™, Pluronic R™, Tetronic™, Pluradot™), alkyl, aryl polyetheralcohol (Tyloxapol™), perfluoroalkyl polyoxylated amides,N,N-bis[3-D-gluconamidopropyl]cholamide, decanoyl-N-methylglucamide,n-decyl α-D-glucopyranozide, n-decyl β-D-glucopyranozide, n-decylβ-D-maltopyranozide, n-dodecyl β-D-glucopyranozide, n-undecylβ-D-glucopyranozide, n-heptyl β-D-glucopyranozide, n-heptylβ-D-thioglucopyranozide, n-hexyl β-D-glucopyranozide, n-nonanoylβ-D-glucopyranozide 1-monooleyl-rac-glycerol,nonanoyl-N-methylglucarnide, n-dodecyl α-D-maltoside, n-dodecylβ-D-maltoside, N,N-bis[3-gluconamidepropyl]-deoxycholamide, diethyleneglycol monopentyl ether, digitonin, heptanoyl-N-methylglucamide,heptanoyl-N-methylglucamide, octanoyl-N-methylglucamide, n-octylβ-D-glucopyranozide, n-octyl α-D-glucopyranozide, n-octylβ-D-thiogalactopyranozide, n-octyl β-D-thioglucopyranozide.

Zwitterionic surfactants include but are not limited to betaine(R₁R₂R₃N⁺R′CO₂ ⁻, where R₁R₂R₃R′ are hydrocarbon chains and R₁ is thelongest one), sulfobetaine (R₁R₂R₃N⁺R′SO₃ ⁻), phospholipids (e.g.,dialkyl phosphatidylcholine),3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate,N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate,N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-octyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and dialkylphosphatidyl-ethanolamine.

The polynucleotide compositions of the invention can be administeredorally, topically, rectally, vaginally, by pulmonary route by use of anaerosol, or parenterally, i.e. intramuscularly, subcutaneously,intraperitonealily or intravenously. The polynucleotide compositions canbe administered alone, or it can be combined with apharmaceutically-acceptable carrier or excipient according to standardpharmaceutical practice. For the oral mode of administration, thepolynucleotide compositions can be used in the form of tablets,capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutionsand suspensions, and the like. In the case of tablets, carriers that canbe used include lactose, sodium citrate and salts of phosphoric acid.Various disintegrants such as starch, and lubricating agents such asmagnesium stearate, sodium lauryl sulfate and talc, are commonly used intablets. For oral administration in capsule form, useful diluents arelactose and high molecular weight polyethylene glycols. When aqueoussuspensions are required for oral use, the polynucleotide compositionscan be combined with emulsifying and suspending agents. If desired,certain sweetening and/or flavoring agents can be added. For parenteraladministration, sterile solutions of the conjugate are usually prepared,and the pH of the solutions are suitably adjusted and buffered. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic. For ocular administration, ointmentsor droppable liquids may be delivered by ocular delivery systems knownto the art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. For pulmonary administration,diluents and/or carriers are selected to be appropriate to allow theformation of an aerosol.

The following examples will serve to further typify the nature of theinvention but should not be construed as a limitation on the scopethereof, which is defined solely by the appended claims.

EXAMPLE 1 Transfection Efficiencies—First Embodiment Complex

This experiment sought to introduce plasmid pβ-Gal into NIH 3T3 cells, amouse mammary tumor cell line. Plasmid pβ-Gal comprises plasmid pUC19(available from the Institute of Gene Biology, Russian Academy ofSciences) into which a hybrid of a eukaryotic transcription unit and aE. coli β-galactosidase has been incorporated. With this plasmid, theefficiency of cell uptake can be measured by measuring β-galactosidaseactivity extractable from the treated cells. The copolymer utilized wasa triblock copolymer of formula (XIV) wherein x plus z was 51 and y was39 (hereinafter “Pluronic A”). The polycation utilized waspoly(N-ethyl-4-vinylpyridinium bromide) (“EVP-Br”). A 10 μg/ml solutionof pβ-Gal (predominantly supercoiled) was prepared in a solution of PBScontaining 10 mg/ml of pluronic A and 45, μg/ml of pEVP-Br. Theseamounts there calculated to provide a ratio of polycation basic groupsto plasmid phosphate groups of about 10. The ratio of pluronic A to DNAwas about 10⁴. This stock preparation was filter sterilized and aportion was diluted ten fold with serum-free Dulbecco's Modified Eagle'sMedium (“DMEM”), so that the concentration of pβ-Gal was 1 μg/ml. Thissolution was the “Pluronic A transfecting medium.”

The NIH 3T3 cells were grown in monolayer culture at 37° C. under 5%CO₂, utilizing a DMEM medium containing 2 mM glutamine and 10% fetalcalf serum (“FCS”). Cells were grown in monolayer culture were scrapedand prepared for the transaction process by washing three times withfresh medium.

Aliquots of washed cells that were to be transformed by the method ofthe invention were suspended at a concentration of 10⁶ cells/ml inPluronic A transfecting medium. The suspended cells were incubated for 2hours at 37° C. and under 5% CO₂. The cells were then washed with freshmedium and re-plated.

Aliquots of cells that were to be transfected by calcium phosphateprecipitation were transfected as recommended by Promega of Madison,Wis., in their manuscript Profection Mammalian Transfection Systems,Technical Manual, 1990. Specifically, pβ-Gal was mixed with 0.25M CaCl₂The mixture was mixed with an equal volume of 2×HBS (Hanks Buffer Salt,available from GIBCO, Grand Island, N.Y.) to create a mixture containing1 μg/mL pβ-Gal. The opaque mixture was incubated at room temperature for10 minutes and then applied to the cells. The suspended cells wereincubated for 2 hours at 37° C. and under 5% CO₂. The cells were thenwashed with fresh medium and re-plated.

The repeated cells were incubated for 48 hours in DMEM medium containing10% FCS. During the incubation, the medium was replaced with freshmedium at 16 hours. After the 48 hour incubation, the cells for eachincubation were collected by scrapping, washed with PBS, and resuspendedin 100 μl of 0.2 M Tris-HCL (pH 7.4). The cells were lysed with severalfreeze/thaw cycles, and centrifuged at an excess of 6,000×/g. 50 μl ofsupernatant was removed from each lysate tube and mixed with 50 μl of asolution of 0.1 mM 4-methyl-umbelliferril-β-D-galactopiraniside (thesubstrate), 0.1 M sodium phosphate (pH 7.4). Each mixture was incubatedfor 20 min. at 37° C. to allow any 9-galactosidase present to act on thesubstrate. 50 μl of 0.4 M glycine, pH 10.5, was added to terminate theβ-galactosidase reaction. β-galactosidase activity was indicated by thepresence of methylbelliferon, which can be measured by fluorescencespectroscopy (λ_(ex)=365 nm, λ=450 nm). The results were as follows:

Treatment Relative Enzyme Activity ± SEM (n = 4) Pluronic A 320 ± 42Calcium Phosphate 17 ± 5 Precipitation

EXAMPLE 2 Transfection Efficiencies—First Embodiment Complex

In these experiments, transfection efficiencies with MDCK cells (derivedfrom canine kidney) were examined. Again, pβ-Gal was the indicatorpolynucleotide. The polycation component of the polynucleotide compriseda copolymer of N-ethyl-4-vinylpyridinium bromide andN-cetyl-4-vinylpyridinium bromide, the monomers incorporated in a molarratio of 97:3, respectively (hereinafter “pEVP-co-pCVP-Br”). The blockcopolymer comprised a triblock copolymer of formula (XIV) wherein x+zwas 18 and y was 23 (hereinafter “Pluronic B”). A Pluronic Btransfecting solution of 1 μg/ml pβ-Gal, 3 μg/ml PEVPco-pCVP-Br, and 1%(w/v) Pluronic B was prepared in Example 1. The ratio of polycationbasic groups to nucleotide Phosphates was about 7. The weight ratio ofPluronic B to pβ-Gal was about 5×10³.

MDCK cells were plated at 8-10⁵ cells per plate onto 90 mm plates andincubated overnight under serum-containing growth medium. The serumcontaining medium was then replaced with serum-free medium, and thecells were incubated at 37° C., under 5% CO² for 24 hours. For the cellsto be treated with polynucleotide complex, the medium was then replacedwith 5 ml Pluronic B transfecting solution. The cells were incubated,with gentle rocking, at 37° C., under 5% CO₂. In control experiments,cells were transfected with polynucleotide complex, the medium was thenreplaced with 5 ml Pluronic B transfecting solution. The cells wereincubated, with gentle rocking, at 37° C., under 5% CO₂, for 2 hours. Incontrol experiments, cells were transfected using the calcium phosphateprocedure as described above (except that plated cells, not suspendedcells, were transfected).

After treatment with Pluronic B transfecting solution or calciumphosphate, the cells were washed 5-6 times with fresh medium. They werethen incubated in DMEM containing 10% FCS for 48 hours at 37° C., under5% CO₂. After the first 16 hours of this incubation, the medium wasreplaced. After the incubation, the cells were washed with PBS, releasedfrom their plates by trypsinization, and again washed with PBS.β-Galactosidase was measured as described for Example 1. The resultswere as follows:

Relative β-galactosidase activity ± Treatment SEM (n = 4) Pluronic B 910± 45 Calcium Phosphate Precipitation  81 ± 17

EXAMPLE 3 Transfection Experiments—First Embodiment Complex

In these experiments, transfection efficiencies with Chinese hamsterovary (CHO) cells were examined. The polynucleotic component of thepolynucleotic complex was pβ-Gal. The polycation component comprisedpEVPBr. The block copolymer comprised an octablock copolymer formula(XVII), wherein i was equal to 10 and j was equal to 12 (hereinafter“Pluronic C” available from BASF). A Pluronic C transfecting solution of1 μg/ml pβ-Gal, 4μg/ml pEVP-Br, and 1% (w/v) Pluronic C was prepared asin Example 1. The ratio of basic groups to nucleotide phosphates was 10.The weight ratio of Pluronic C to pβ-Gal was 10³. The transfectionprotocol was t he same as that used in Example 2. The results were asfollows:

Relative β-galactosidase Treatment activity ± SEM (n = 4) Pluronic B 910± 45 Calcium Phosphate Precipitation  81 ± 17

EXAMPLE 4 Bacterial Transformation—Second Embodiment Complex

In these experiments, transformation efficiencies using the MC5 strainof Bacillus subtilis were examined. The polynucleotide component of thepolynucleotide complex was plasmid pBC16, a plasmid encodingtetracycline resistance. A block copolymer according to formula (VI) wasused. In particular, the block copolymer was apoly(oxyethylene)-oly((N-ethyl-4-vinylpyridinium bromide) of formula(XXI), wherein i was 44, and j was 20. A stock solution of secondembodiment polynucleotide complex was prepared consistent with thetransfection solutions described above. The ratio of copolymer basicgroups to DNA phosphates in the solution was 0.2. Bacteria weresuspended in Spizizen 11, a transformation media (see, Spizizen,F.N.A.S., U.S.A. 44:1072 (1958)), and aliquots of cells were incubatedin varying concentrations of either polynucleotide complex or freepBC16. The cells were incubated with complex or free DNA for one hour at37° C. Following the incubation, the cells were plated onto agar mediacontaining 10 mg/ml tetracycline. The results, measured by the number oftetracycline-resistant colonies produced under each of the experimentalconditions, were as follows:

DNA concentration Transformation (10⁶ clones/ng DNA) (ng/ml)Polynucleotide Complex Free Polynucleotide 5 300 (±15) 0 10 450 (±22) 3(±1) 20 400 (±26) 3 (±4) 50 220 (±17) 20 (±5) 

EXAMPLE 5 Protection from Nuclease

For this example, a complex of plasmid pTZ19 and a diblock copolymer offormula (XXI) (poly(oxyethylene)-poly((N-ethyl-4vinylpyridiniumnbromide), wherein i was 44 and j was 20) was formed. The solution ofpolynucleotide complex dissolved in PBS contained about 4 μg/ml ofplasmid and 20 μg/ml of diblock copolymer. These amounts resulted in aratio of base groups in the polycation block to DNA phosphate groups of5. For control incubations, an equivalent amount of free plasmid wasdissolved in buffer. PVUII nuclease was added to solution samplescontaining free DNA or polynucleotide complex, and the amount ofundigested, circular plasmid DNA, after various digestion times, wasdetermined by electrophoresis in a polyacrylamide gel. See Kabanov etal., Biopolymers, 31:1437-1443 (1991). The results were as follows:

Circular DNA (% of initial) Time of Incubation Complex Free DNA 0 100100 5 100 20. 10 100 8 30 100 4 60 100 1 180 100 0 600 100 0

EXAMPLE 6 Oligonucleotide Stabilization

For this example, a complex containing an oligonucleotide complementaryto the transcription initiation site of the HIV-1 tat gene (“anti-tat”,comprising GGCTCCATTTCTTGCTC) was prepared using the diblock copolymerof formula (XIX) (polyoxyethylene-poly(L-alanine-L-lysine), wherein i is44 and j is 8). The oligonucleotide complex was prepared in PBS Buffer(pH 7.0) at a concentration of 0.75 OD₂₆₀/μl oligonucleotide. The ratioof polyeation imino and amino groups to polynucleotide phosphate groupswas about 50. The mixture was incubated for one hour at room temperatureto allow for the formation of the complex. Then, the complex waspurified by gel filtration chromatography on Sephadex G-25 using 0.05 MNaCl as the eluent. The resulting solution of complex exhibited aconcentration of 0.11 OD₂₆₀/μl of oligonucleotide. A comparable solutionof uncomplex oligonucleotide was prepared. An aliquot of murine bloodplasma (10 μl) was mixed with an equal volume of oligonucleotide complexsolution or a solution of free oligonucleotide. Samples were incubatedat 37° C. for various time periods. To stop the reaction of theoligonucleotides with enzymes in the plasma, the samples were dilutedwith water and extracted with a water-saturated mixture ofphenol:chloroform (1:1). The aqueous phase of the extraction wasisolated, and the oligonucleotide therein was precipitated with 3%lithium Perchlorate. The precipitate was washed with acetone, and thendissolved in 100 μl of water. The presence of undergradedoligonucleotide was determined by high performance liquid chromatographyusing a C₁₈-Silasorb column (4×90 mm, Gilson, France) and a gradient ofacetonitrile in 0.05 M triethylammoniumacetate (pH 7.0) as the eluent.The results were as follows:

Time of Undergraded oligonucleotide (%) Incubation Complex Free Oligo 0100 100 3 hours 88 28 6 hours 70 17 24 hours  36 0

EXAMPLE 7 Oligonucleotide Stabilization

This example examined the stability of the oligonucleotide described inExample 6, when complexed with a diblock copolymer of formula (XX)(polyoxyethylene-polypropyleneimine/butyleneimine, wherein i is 44 and jis 4-8) was examined. The same methodologies that were applied inExample 6 were applied for this example, except that the oligonucleotideconcentration was about 0.13 OD₂₆₀/μl. The results were as follows:

Time of Undergraded oligonucleotide (%) Incubation Complex Free Oligo 0100 100 3 hours 70 28 6 hours 57 17 24 hours  28 0

EXAMPLE 8 Antisense Cell Incorporation Efficiencies

This experiment examined the effectiveness of “anti-MDR”, an antisensemolecule comprising a 17-chain oligonucleotide of sequenceCCTTCAAGATCCATCCC complementary to positions 422-438 of the mRNAencoding the MDRI gene product, in reversing multi-drug resistance inSKVLB cells. SKVLB cells are multi-drug resistant cells derived from aovarian cancer cell line. The MDR1 gene has been identified asresponsible for the multi-drug resistance in SKVLB cells. Endicott andLing, Ann. Rev. Biochem., 58:137 (1989). In particular, the efficiencyof the anti-MDR oligonucleotide in the polynucleotide complex of theinvention and when in the free state was cornpared. As controls, thefree and completed form of the anti-tat oligonucleotide described abovewere also used. The polynucleotide complexes were formed with thediblock copolymer of formula (XX)(polyoxyethylene-polypropyleneimine/butyleneimine, where i was 44 and jwas 9-10). The; complexes were prepared by the procedures described inExample 6. The oligonucleotide concentration in the complex or in thefree state was 0.17 OD₂₆₀/μl. The copolymer was present in theconcentration sufficient to define a ratio of polycation segment iminoand amino groups to oligonucleotide phosphate groups of 10.

The SKVLB cells were incubated for 3 days at 37° C. under 5% CO₂ in thepresence of free or completed oligonucleotide, (at a concentration of 20μM based on oligonucleotide content). Fresh media including free orcompleted oligonucleotide was added every 12 hours.

The daunomycin cytotoxicity (IC₅₀) with respect to the cells treated asdescribed above was measured using the method of Alley et. al., CancerRes., 48:589-601. The results were as follows:

Treatment of Cells Daunomycin IC₅₀ (ng/ml) (n = 4) Control (untreatedcells) 8.0 Anti-MDR Complex 0.3 Anti-tat Complex 8.2 Free Anti-MDR 2.1Free Anti-tat 7.9

EXAMPLE 9 Antisense Oligonucleotide Designed to Inhibit Herpes Virus

This experiment utilized a 12-chain oligonucleotide, which had beencovalently modified at its 5′ end with undecylphosphate substituent andat is 3′ end with a acridine group, was used. This oligonucleotidemodification has been described by Cho-Chung et. al., Biochemistry Int.,25:767-773 (1991). The oligonucleotide sequence utilized, CGTTCCTCCTGU,was complementary to the splicing site at 983-994 of the Herpes SimplexVirus 1 (“HSV-1”). As a control, an equivalently modified sequence(AGCAAAAGCAGG) complementary to the RNA produced by influenza virus wasutilized. The oligonucleotides were applied to HSV-1 infected cells ineither the complexed or the free state. When a complex was utilized, thecomplex was formed with the diblock copolymer of formula (XIX)(polyoxyethylene-poly(L-alanine-L-lysine), wherein i was equal to 44 andj was equal to 8). Oligonucleotide complexes were formed as described inExample 6.

African marmoset kidney cells (“Vero” cells) were infected with HSV-1virus (strain L2, obtained from the Museum of Virus Strains, D. I.Ivanovskii, Inst. of Virol., Russian Federation), as described byVinogradov et al., BBRC, 203:959 (1994). The infected cells were washedwith PBS. After washing, fresh RPMI-L 640 media containing 10% of fetalcalf serum and free or complex oligonucleotide was added to the cell.The cells were then incubated at 37° C. under 5% CO₂ for 24 hours. TheHSV-1 infectivity of the of the cell media was then determined using thepatch production method described by Virology, A Practical Approach,Mahy, Ed., IRL Press, Washington, D.C., 1985. The results, utilizingvarying concentrations of oligonucleotide, were as follows:

Oligo Conc. HSV-1 Infectious Titre (CPE₅₀/ml) (n = 7) Treatment 0.2 μM1.0 μM 5.0 μM Control 1.0 (±0.5) × 10⁶ 1.0 (±0.5) × 10⁶ 1.0 (±0.5) × 10⁶(untreated infected cells) Anti-HSV 1.4 (±0.2) × 10² 0.5 (±0.3) × 10² 0complex Anti-influenza 1.0 (±0.6) × 10⁶ 0.7 (±0.1) × 10⁶ 0.8 (±0.2) ×10⁶ complex Free Anti-HSV 0.9 (±0.4) × 10⁵ 2.3 (±0.7) × 10³ 1.6 (±0.4) ×10² Free Anti- 1.1 (±0.4) × 10⁶ 0.9 (±0.2) × 10⁶ 0.6 (±0.3) × 10⁶Influenza

EXAMPLE 10 Antisense Oligonucleotide Designed to Inhibit Herpes Virus

Unless otherwise noted, this example utilized the same procedures aswere utilized in Example 9. The cells utilized were BHK cells, a Chinesehamster kidney cell line. When the complexed form of theoligonucleotides was used, the complex was formed with the diblockcopolymer of formula (XVII) (6olyoxyethylene-poly-L-lysine, wherein iwas 44 and j was 30), using the procedure described in Example 6. Theconcentration of the stock solution of complex was 0.09 OD₂₆₀/μl. Theratio of polycation segment imino and amino groups to oligonucleotidephosphates was 10. The oligonucleotides, in complexed or free form, wereapplied to the cells at a concentration of 3.0 μM. The results were asfollows:

Treatment of cells HSV-1 infectious titre (CPE₅₀/ml) n = 7 Control(untreated infected cells) 10 (±3) × 10³ Anti-HSV complex 8 (±6)Anti-influenza complex 13 (±4) × 10³ Free Anti-HSV 50 (±14) × 10² FreeAnti-influenza  9 (±2) × 10³

EXAMPLE 11 In Vivo Inhibition of HSV

Polynucleotide complexes between the block copolymer of formula (XVII)(polyoxyethylene-poly-L-lysine, wherein i was 44 and j was 30) and theAnti-HSV and Anti-Influenza oligonucleotides were formed using themethods outlined in Example 9. The concentration of the stock solutionsof complexes was 0.9 OD₂₆₀/μl. The ratio of polycation segment imino andamino groups to oligonucleotide phosphates was 10.

Inbred white mice (body weight: 6 to 7 g) were infected with HSV-1(strain Cl from Belorussian Res. Inst. of Epidemiol. & Microbiol.,Minsk) by intraperitoneal injection of 30 μl of a virus suspension(titre: 10⁻⁷ LD₅₀/ml).

Either Anti-HSV complex, Anti-influenza complex, free Anti-HSV or freeAnti-Influenza were injected (10 μl) into the tail vein of a given mouseat each of 2, 12, 24, 48 or 72 hours post-infection. The results were asfollows:

Survived animals/Amount of Animals in a group Treatment of mice Exp. 1Exp. 2 Exp. 3 % Survival Control (infected mice) 1/9 1/10 2/10 13.7Anti-HSV complex 8/9 6/10 7/10 73.0 Anti-influenza complex  2/10 0/101/10 10.0 Free Anti-HSV  1/10 1/10 0/10 7.0 Free Anti-influenza 0/9 1/100/10 7.0

EXAMPLE 12 Plasma Life of Polynucleotide Complex

A ³²P-labelled 17-mer (GGCTCCATTTCTTGCTC) complementary to thetranscription initiation site of the HIV-1 tat gene was utilized in thisexample. The oligonucleotide was modified at its 5′-end with cholesterolas described by Boutorin et al., Bioconjugate Chemistry, 2:350-356(1990). A polynucleotide conjugate of the oligonucleotide was formedwith the block copolymer of formula (XX)polyoxyethylene-poly(propyleneimine/butyleneimine), wherein i was 44 andj was 9 to 10). The concentration of the stock solution (dissolved inPBS) of complex was 0.18 OD₂₆₀/μl. The ratio of polycation segment iminoand amino groups to oligonucleotide phosphates was 50.

Male C57/Bln/6 mice (weight: 20-24 g; obtained from the Russian ResearchCenter of Molecular Diagnostics and Therapy, Moscow) received 50 μlintravenous injections of Anti-HIV conjugate or free Anti-HIV, at 0.18OD₂₆₀/μl dissolved in PBS. At defined times after the injections, bloodsample were taken from the tail vein and the animals were sacrificed.The amount of radioactive material in blood or tissue sample wasdetermined by liquid scintillation counting (after appropriatesolubilizations). The results were as follows:

Plasma levels (% of Liver levels Liver levels injected dose) (% of (% ofTime after Anti-HIV Free Anti- injected dose) injected dose) injection(min) Conjugate HIV Prep. A Prep. B 0 100 100 0 0 5 95 58 3 7 10 91 40 519 15 84 33 7 26 20 79 27 9 30 30 75 20 10 35

EXAMPLE 13 Cationic Block Copolymer Synthesis

1,4-dibromobutane (5.4 g, 25 mmoles, from Aldrich Co., Milwaukee, Wis.)was added to a solution of N-(3-aminiopropyl)-1,3-propanediamine (6.55g, 50 mmoles, from Aldrich Co.) dissolved in 100 ml of 1,4-dioxane. Thisreaction mixture was stirred at 20° C. for 16 h. The product of thisreaction spontaneously precipitates from solution as the hydrobromidesalt. This precipitated first intermediate was collected and twice driedby rota-evaporation from a solution of 10% triethylamine in methanol.This evaporation procedure was effective to remove substantial amountsof the bromide salt. The first intermediate was dissolved in 50 ml of1,4-dioxane and reacted with 2.7 g (12.5 mmoles) of 1,4-dibromobutane.Again, the reaction proceeded for 16 h at 20° C., and the resultingsecond intermediate was recovered and dried as above.

The second intermediate was neutralized with acetic acid to a pH of 7-8and purified by gel filtration on Sephadex G-25, using an aqueouseluent. Three major polymine fractions were obtained, having apparentmolecular weights of 1060, 700 and 500, respectively.

Poly(oxyethyleneglycol) (1.5 g, M.W. 1500, from Fluka) was dissolved in8 ml of 1,4-dioxane and reacted with 0.17 g (1 mmole) ofN,N′-carbonylimidazole (Aldrich Co.) at 20° C. for 3 h. The reactionmixture was divided into two parts. Each part was mixed with 4 ml of a10% (w/v) solution of either the 1060 or 700 MW polyimine fraction,which solution further contained 0.01 N NaOH. The mixture was stirredfor 16 h at 20° C. From this mixture, block copolymers of formula (XX)and various MW ranges were isolated by gel filtration.

EXAMPLE 14 Cationic Block Copolymer Synthesis

0.5 g of a succinimidyl carbonate of methoxy-POLY(ETHYLENE GLYCOL) (MW5000, Shearwater Polymers, Inc., USA) was dissolved in 1,4-dioxane. Thisdioxane solution was added to an aqueous solution containing 0.2 g ofthe 1060 MW polyimine polymer described above, which aqueous solutionfurther included.

0.01 N NaOH. This reaction mixture was stirred at 20° C. for 16 h. Apolymer of formula (XXII) was isolated from the reaction by gelfiltration.

EXAMPLE 15 Cationic Block Copolymer Synthesis

1.5 g of poly(oxyethyleneglylol) (MW 8000, Fluka) were dissolved in 8 mlof 1,4-dioxane. 0.34 g (2 mmole) of N,N′-carbonylimidazole (Aldrich Co.)were added to the solution and reacted for 3 h at 20° C. 8 ml of anaqueous solution containing 0.01 N NaOH and 15% (w/v) of the 500 MWpolyimine polymer described above in Example 13 was then added to thefirst reaction mixture. The resulting mixture was reacted for 16 h at20° C. with stirring. A polymer of formula (XXIII) was isolated from thesecond reaction mixture by gel filtration.

EXAMPLE 16 Conjugate Synthesis with Oligonucleotide

A 12-mer oligonucleotide, 5′-CGTTCCTCCTGU (“Oligo A”) complimentary tothe splicing site (positions 983-994 on the viral genome) of the earlymRNA of type 1 Herpes Simplex Virus (“HSV-1”), was synthesized using a380B-02 DNA-synthesizer (Applied Biosystems, CA). The synthesizer usedphosporamidite chemistry and an 8 min. synthesis cycle. Cycle conditionsand preparation of the crude product were done as recommended by AppliedBiosystems. The crude Oligo A obtained from the synthesis wasprecipitated from a 1 M LiCl solution (0.5 ml) with acetone (2 ml). Theprecipitate was dissolved in triethylammonium acetate buffer andpurified by reverse-phase high performance liquid chromatography on aSilasorb C18 column (9×250 mm, Gilson, France) developed with anacetonitrile gradient in a 20 mM TEAA buffer (pH 8.5).

The 3′-terminal of the purified Oligo A was oxidized with periodate tocreate an aldehyde and conjugated by reductive alkylation with ahexamethylene-diamine linker, creating an amine derivative. SeeChe-Chung et al., Biochem. Intemat., 25:767 (1991); Vinogradov et al.,BBRC, 203:959 (1994). “Pluronic A”, a block copolymer of formula (XIV)(x=25, y+38, z=25) was similarly oxidized to create terminal aldehydes.The amine derivative (1 mg) was dissolved in 100 μl of 0.1 M boratebuffer (pH 9.0) and mixed with 2 mg of the Pluronic A derivative. 1.5 mgof sodium cyanoborohydride was added to the mixture to reduce theSchiffs bases formed between the amine and aldehyde groups. Thisreaction was allowed to proceed for 12 hours at 4° C. The polymericproduct of this reaction was isolated by gel filtration chromatographyon Sephadex LH-20, utilizing 90% aqueous isopropanol as the eluent. Theconjugate so obtained is referred to hereinafter as “Oligo A Conjugate.”

EXAMPLE 17 The Effect of Oligo A Conjugate on Virus Production

Oligo A and Oligo A Conjugate were separately dissolved in RPMI 1640medium (ICN Biomedicals Inc., Costa Mesa, Calif.) to a finalconcentration of 0.2 mM (based on oligonucleotide absorbance). Thesestock solutions were then filtered through 0.22 μm filters to remove anypossible bacterial or fungal contamination.

Monolayers of Vero cells were incubated for 1 hour at 37° C. inserum-free RPMI 1640 together with various concentrations of Oligo A orOligo A Conjugate. The monolayers, while still exposed tooligonucleotides, were then infected with 1 plaque forming unit percultured cell of HSV-1, strain L2 (from the Museum of Virus Strains ofthe D.I. Ivanovskii Institute of Virology, Russian Academy of Sciences,Russian Federation). This infection method has been described byVinogradov et al., BBRC, 203:959 (1994). After 8 hours of exposure tovirus and oligonucleotides, the medium on the cells was replaced withfresh medium containing 10% FCS. Medium from the cells was collected at22 and 39 hours after the ineffective incubation, and the virus titer inthe collected medium was determined as described in Virology, APractical Approach, Mahy, Ed., IRL Press, Oxford Univ. Press,Washington, D.C. 1985, The results were as follows:

Infectious Titer of HSV-1 (PFU/ml) Sample concentration Oligonucleotide22 hours past 39 hours past (mM) concentration (μM) infection infectionControl (cells without 0 5 × 10⁶ 1 × 10⁷ oligonucleotides) Oligo A 10 3× 10⁶ 5 × 10⁶ 5 5 × 10⁶ 1 × 10⁷ 2 5 × 10⁶ 1 × 10⁷ 1 5 × 10⁶ 1 × 10⁷Oligo A Conjugate 10 0 0 5 0 5 × 10² 2 1 × 10³ 7 × 10³ 1 5 × 10⁴ 3 × 10⁶

EXAMPLE 18 Synthesis of a Phosphonate Monomer

40 mmoles of butanediol-1,3 (Merck) dissolved in 50 ml of anhydrouspyridine (Aldrich) were reacted with 20 mmoles4,4′-dimethoxytritylchloride (Sigma) for 1.5 hours at 20° C. Thereaction was monitored using thin layer chromatography on the silicagelplates (Merck) developed with a chloroform:methanol (95:5). The Rf ofthe product was 0.6. The reaction mixture was added to 200 ml of an 8%aqueous solution of the sodium bicarbonate and the product extractedwith chloroform. The chloroform extract was evaporated in vacuum and theresulting oily first intermediate was used in the next stage of thesynthesis.

12 mmoles of first intermediate were dissolved in 30 ml of anhydrous1,4-dioxane, containing 3.14 ml (18 mmoles) of diisopropylethylamine(Aldrich). 18 mmoles of salicylchlorophosphite (Sigma) dissolved in 10ml of ahydrous 1,4-dioxane were added to the diisopropyethylaminesolution in small portions under an inert, argon atmosphere. Thereaction mixture was incubated during 1 hour at 20° C. The reaction wasmonitored by the thin layer chromatography as described above. The Rf ofthe product was 0.05. 10 mls of water were added to the reactionmixture. After 30 min., the solvent was evaporated. The product wasdissolved in 100 ml of chloroform and the solution obtained was washedstepwise with (1) 100 ml of 8% aqueous solution of the sodiumbicarbonate, (2) 100 ml of 0.2 M triethyammoniumacelate solution (pH7.2), and (3) 100 ml of water. The organic solvent was evaporated andthe oily remainder, containing the phosphonate monomer was purified bychromatography on silicagel column, using stepwise gradient of (1)chloroform, (2) 3% methanol in chloroform and (3) 6% methanol inchloroform. The yield of the monomer was 4.1 g (=7.3 mmol, 63%). Theproduct, having structure:

wherein DMT represents a dimethoxytrityl group, can be termed“Phosphonate Monomer A.”

EXAMPLE 19 Synthesis of Polycation BDP

A 0.05 M solution of the phosphonate Monomer A in anhydrouspyridine:acetonitrile mixture (1:1) was placed in the position 6 of theDNA-synthesator (model 380-B02, Applied Biosystems, CA). A 2% solutionof adamantoilchloride (Sigma) in the mixture acetonitrile:pyridine(95:5) was used as a condensing agent. The synthesis was conducted usingthe program modified for an H-phosphonate cycle (Sinha and Striepeke In:Oligonucleotides and Analogues: A Practical Approach, Eckstein Ed. IRLPress, Oxford, N.Y.-Tokyo, p.185, 1991) and the DMT-group was preservedafter the synthesis was complete. Adenosine (4 μmoles) immobilized on astandard CPG-500 solid support was used as a first unit during thepolymer synthesis (Vinogradov et al. BBRC, 203, 959 (1994). Thesynthesizer was programmed to add Phosphonate Monomer A repeating unitsto the adenosine monomer. Following all synthesis steps, theH-phosphonate groups on the immobilized substrate were oxidized with thesolution of 104 mg of hexamethylenediamine (Sigma) in 0.6 ml of amixture of anhydrous pyridine:CCl₄ (5:1) applied for 15 min. at 20° C.,then the carrier was washed with the pyridine:acetonitrile mixture(1:1).

Deblocking and cap removal was achieved by ammonolysis (Oligonucleotidesand Analogues. A Practical Approach, Eckstein Ed. IRL Press, Oxford,N.Y.-Tokyo, 1991). The product was purified by HPLC using Silasorb C,column (9×250 mm. Gilson, France) in the acetonitrile gradient (0-80%).The peak, containing dimethoxytritylated-product was collected, thesolvent was evaporated and the remainder was treated with 80% aceticacid (20 min). The acetic acid was evaporated and the polycation waspurified again by HPLC. The yield of the 15-mer (counted in terms ofPhosphonate Monomer A) is 50% (2.2 μmoles). This created a polymeraccording to formula A. The polymer is termed hereinafter “BDP.”

EXAMPLE 20 Solid Phase Synthesis of the Diblock CopolymerPolyoxyethylene-BDP

Dimethoxytrityl-polyethyleneoxide-H-phosphonate was synthesized asdescribed in Example 18 using polyethyleneglycol (1500 M.W. from Fluka)instead of butanediol-1,3. The BDP polycation was synthesized asdescribed in Example 19, except that, at the last stage of the chaingrowth, dimethoxytrityl-polyethyleneoxide-H-phosphonate was introducedas the last building block. The H-phosphonate groups of the blockcopolymer were oxidized as described in Example 19 usingtetramethylenediamine (Sigma) instead of hexamethylenadiamine, resultingin the formation of phosphonamide bonds between the diamines and thebackbone phosphates.

EXAMPLE 21 Solid Phase Synthesis of the Oligonucleotide-BDP DiblockCopolymer

A diblock copolymer comprising 12-mer oligonucleotide, 5′-GGTTCCTCCTGU(Oligo A, complementary to the splicing site of the early mRNA of type 1Herpes Simplex Virus (HSV-1), Vinogradov et al. BBRC, 203, 959 (1994))and the BDP polymer was synthesized in DNA synthesator. First the BDPpolymer was synthesized as described in Example 19, except that it wasnot removed from the support. Then the oligonucleotide chain wassynthesized step-wise onto BDP polycationic polymer linked to the solidstate support using the standard phosphoroamidite chemistry as describedby Vinogradov et al. BBRC, 203, 959 (1994). The H-phosphonate groups ofthe diblock copolymer were oxidized as described in Example 19 usingtetamethylenediamine (Sigma) instead of hexamethylenediamine.

EXAMPLE 22 Effect of Oligonucleotide-BDP Diblock Copolymer on ViralGrowth

The experiment was performed exactly as described in Example 17 exceptthat (1) the oligonucleotide-BDP copolymer of Example 21 was used and(2) a single concentration of oligonucleotide-BDP copolymer (conjugate)was used (4.4 M).

Sample Virus titre after 39 hours Control (without oligonucleotide) 500× 10⁴ Nonmodified Oligo A 500 × 10⁴ Diblock  5 × 10⁴

EXAMPLE 23 Synthesis of Branched Polyimine Polycation

A. The polyimine polycation (“polyspermine”) was obtained by stepwisepolycondensation of N-(3-aminopropyl)-1,3-propanediamine and1,4-dibromobutane as described in Example 13 and used withoutconjugating to poly(ethylene glycol).

B. The polyimine polycation synthesized in A was modified by dansylchloride to obtain a fluorescent dansyl-labeled substance, purified bythin layer chromatography and a major component of the mixture (over 75%in most batches) was analyzed by electrospray mass-spectrometry inpositive charge mode. The results were compared with mass-spectraobtained for the N-(3-aminopropyl)-1,3-propanediamine modified withdansyl chloride. Dansyl-labeled N-(3-aminopropyl)-1,3-propanediaminegave a four-modal peak at M+1, M+2, M+3 and M+4 (667.6, 668.5, 669.6 and670.5). In the spectrum of the polycondensation products there wereobserved two types of polymodal peaks: M and M+54. For M-peaks twodistinct groups were observed, with M/2H+ and M/H+, equal to 598.5 and1195.6 respectively. This molecular mass was very close to a linearpolycation with 12 nitrogen atoms (1221). M+54 peaks at 1249.8 and 652.5correspond to a polycation with CH₂CH₂CH₂CH₂ cross-links.

C. 1H-NMR spectra were obtained for the samples of the polyiminepolycation synthesized in A and dissolved in DMSO. Three groups ofsignals were observed at 1.40-1.80 ppm (Ha), 1.80-2.20 ppm (Hb) and2.35-2.80 ppm (Hc). Ha related to CH₂CH₂CH₂CH₂ protons, Hb related toCH₂CH₂CH₂ protons, Hc related to —NHCH₂ and protons. Integration ofresonance signals for these three groups gave a ratio Ha:Hb:Hc equal to1.00:0.75:1.20. The theoretical ratio for linear polycations with 12nitrogen atoms is 1.00:1.33:3.67. Increase in Hb:Ha and Hc:Ha ratiossuggested presence of branched structures with a mixture of primary,secondary and tertiary amines.

D. The concentration of primary amino groups in the polyimine polycationsynthesized in A was determined by fluorescamine method as described byWeigele et al., J. Amer. Chem. Soc., 1972, 94:5927. The total amount ofprimary, secondary, and tertiary amino groups in the polycondensationproduct was determined using potentiometric titration. The ratio of thetotal amount of primary, secondary, and tertiary amino groups to theamount primary amino groups equals 2.7. Given the molecular masses ofthe condensation product determined using mass-spectrometry the resultof this experiment suggests considerable branching, i.e. the presence oftertiary amines.

EXAMPLE 24 Synthesis of Linear Polyimine Polycation

Linear polycations of polyimine type are synthesized by condensation ofa diaminoalkyl and bis-aldehyde in the presence of sodiumcyanoborohydride using a modified reductive amination proceduredescribed by Aziz et al., J. Pharmac. Exper. Therapeutics, 1995,274:181. 0.33 g of malonaldehyde bis(dimethyl acetal) was added in 10 mlof 0.5 N HCl and stirred for 1 h at 20° C. to obtain free bis-aldehyde.1.27 g of N,N′-bis[3-aminopropyl]-1,4-butanediamine was added to thissolution and pH was adjusted to 5.0. The mixture was allowed to stay for1 h at 37° C., then 1.27 g of N,N′-bis[3-aminopropyl]-1,4-butanediaminewas added to it and pH was adjusted to 7.0 using sodium carbonatesolution. The reaction mixture was treated with 0.26 g of sodiumcyanoborohydride and left for additional 1 h at 37° C. The finalslightly yellow solution was desalted by gel permeation chromatographyon the Sephadex G-25 column in 10% methanol and first high-molecularweight fractions revealing primary amino groups in ninhydrine test werefreeze-dried. This yields 0.43 g of the following polyimine polycation:

H[NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃]_(x)NH₂

EXAMPLE 25 Synthesis of Cationic Block Copolymer

1.5 g of poly(ethylene glycol), methyl ester, mw. 5000 Mw. (Sigma) wasactivated by 0.25 g of 1,1′-carbonyldiimidazole in 10 ml of anhydrousacetonitrile for 3 hrs at room temperature. The solvent was evaporatedin vacuo, the residue redissolved in water and dialyzed throughMembra-Cel MD-25-03.5 membrane with cutoff 3500 Da against water.Desalted solution was concentrated in vacuo and used in a reaction with2-fold excess of poly-L-lysine, Mw. 4000, in methanol-water solution for16-24 hrs at room temperature. The conjugate obtained was purified bygel-permeation column chromatography on Sephadex-50 (fine) (Pharmacia)in water and then by reverse phase chromatography on semi-preparativecolumn (Vydac C 18 5 u,10 mm×25 cm) in acetonitrile concentrationgradient. The yield was 70%. Content of amino groups was measured byfluorescamine method and total nitrogen content was determined byelemental analysis to assess the purity of the conjugates. Usually itwas about 75-90% based on graviometry.

EXAMPLE 26 Synthesis of Cationic Block Copolymer

Following the procedure of Example 25 but substituting the 2-fold excessof poly-L-lysine by the same excess of polyethyleneimine,(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y), Mw. 2000 (Aldrich Co.) there isobtained 0.4 g of the cationic diblock copolymer:

CH₃O(CH₂CH₂O)₁₁₄C(O)(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

EXAMPLE 27 Synthesis of Grafted Copolymer

A. 24 g (3 mmol) of poly(ethylene glycol), mw 8000 (Aldrich Co.) weredried by co-evaporation with anhydrous pyridine in vacuo and dissolvedin 50 ml of anhydrous acetonitrile. Then 0.51 g (1.5 mmol) of4,4′-dimethoxytrityl chloride in 30 ml of anhydrous pyridine was addedto this solution dropwise under continuous stirring during 30 min. Themixture was allowed to stand for additional 2 h at room temperature,then the solvents were evaporated in vacuo. The residue was dissolved in50 ml of dichloromethane, extracted with 5% sodium bicarbonate (2×30ml), and applied on the Silicagel column (3×45 cm, 40-60 μm). Stepwiseelution with dichloromethane-methanol solutions separated a slightlyyellow mono-4,4′-dimethoxytrityl-derivative of poly(ethylene glycol)with an yield about 75-85%. The side product of the reaction (10-15%yield) was the bis-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol).

B. 1.5 g of mono-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol) obtained in A was activated by 0.25 g of1,1′-carbonyldiimidazole in 10 ml of anhydrous acetonitrile for 3 hrs atroom temperature. The solvent was evaporated in vacuo, the residueredissolved in water and dialyzed through Membra-Cel MD-25-03.5 membranewith cutoff 3500 Da against water. Desalted solution was concentrated invacuo and then reacted with poly-L-lysine, Mw. 19000 in methanol-watersolution for 24 h at room temperature at a molar ratio of poly(ethyleneglycol) to free amino groups of poly-L-lysine 0.7:1.0. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia) in water and then by reverse phasechromatography on semi-preparative column (Vydac C18 5 u, 10 mm×25 cm)in acetonitrile concentration gradient. This yields a grafted polylysinecopolymer at 35% yield in which 50% of free amino groups are substitutedwith poly(ethylene glycol) as determined by fluorescamine method.

EXAMPLE 28 Synthesis of Grafted Copolymer

A. 24 g (3 mmol) of poly(ethylene glycol), mw 8000 (Aldrich Co.) weredried by co-evaporation with anhydrous pyridine in vacuo and dissolvedin 50 ml of anhydrous acetonitrile. Then 0.51 g (1.5 mmol) of4,4′-dimethoxytrityl chloride in 30 ml of anhydrous pyridine was addedto this solution dropwise under continuous stirring during 30 min. Themixture was allowed to stand for additional 2 h at room temperature,then the solvents were evaporated in vacuo. The residue was dissolved in50 ml of dichloromethane, extracted with 5% sodium bicarbonate (2×30ml), and applied on the Silicagel column (3×45 cm, 40-60 μm). Stepwiseelution with dichloromethane-methanol solutions separated a slightlyyellow mono-4,4′-dimethoxytrityl-derivative of poly(ethylene glycol)with an yield about 75-85%. The side product of the reaction (10-15%yield) was the bis4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol).

B. 1.5 g of mono-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol) obtained in A was activated by 0.25 g of 1,1-carbonyldiimidazolein 10 ml of anhydrous acetonitrile for 3 hrs at room temperature. Thesolvent was evaporated in vacuo, the residue redissolved in water anddialyzed through Membra-Celi MD-25-03.5 membrane with cutoff 3500 Daagainst water. Desalted solution was concentrated in vacuo and thenreacted with polyethyleneimine, Mw. 25,000 in methanol-water solutionfor 24 h at room temperature at a molar ratio of poly(ethylene glycol)to free amino groups of polyethyleneimine 0.7:1.0. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia) in water and then by reverse phasechromatography on semi-preparative column (Vydac C18 5 μm, 10 mm×25 cm)in acetonitrile concentration gradient. This yields a graftedpolyethyleneimine block copolymer at 85% in which 45% of free aminogroups are substituted with poly(ethylene glycol) as determined byfluorescamine method as described by Weigele et al. (J. Amer. Chem.Soc., 1972, 94:5927).

EXAMPLE 29 Synthesis of Grafted Copolymer

Following the procedure of Example 28 but using a molar ratio ofactivated poly(ethylene glycol) to free amino groups ofpolyethyleneimine 0.3:1.0, there is obtained in 80% yield a graftedpolyethyleneimine copolymer in which 24% of free amino groups aresubstituted with poly(ethylene glycol).

EXAMPLE 30 Synthesis of Cationic Block Copolymer

Following the procedure of Example 26 but substituting 6.0 g ofpolyethyleneglycol, mw 20,000 for the excess of polyethylene glycol, mw5,000 there is obtained 6.0 g of the cationic block copolymer:

CH₃O(CH₂CH₂O)₄₅₆C(O)(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

EXAMPLE 31 Synthesis of Cationic Block Copolymer

A. Following the procedure of Example 26 but substituting 1.5 g ofpolyethyleneglycol, Mw. 5,000 by 2.4 g of polyethyleneglycol, Mw. 5,000(Aldrich Co.) there is obtained 1.2 g of the cationic block copolymercontaining polyethyleneinmine and polyethyleneglycol chain segments.

B. The molecular mass of this block-copolymer was determined by staticlight scattering method using DAWN multi-angle laser photometer (WyattTechnology, Santa Barbara, Calif.) which operated at 15 angles andequipped with He—Ne laser (632.8 nm). The samples of the block copolymerwere dialyzed through membrane with cutoff 3,500 Da against 4.5×10⁻³g/ml NaCl and then filtered directly into flow cell used for lightscattering experiments. Weigh-average molecular mass was calculated onthe base of four measurements. Cell constant was determined bycalibration with different concentrations of NaCl. Specific refractiveindex increment (dn/dc) was measured using Wyatt/Optilab 903interferometric refractometer at 632.8 nm. The molecular mass of thesample obtained was 16,000, suggesting that this polymer containedapproximately one polyethyleneinmine segment and two polyethyleneglycolsegments.

C. The number of the primary amino groups in the synthesized sample ofthe copolymer was determined using a modified procedure described byWeigele et al. (J.Amer.Chem.Soc., 1972, 94:5927). To 1.5 ml of a samplein 20 mM sodium borate, pH 9.5 (amino groups concentration up to 100 uM)0.25 ml of fluorescamine solution (0.024%, Sigma) in acetone was addedand vortexed for 5 min. The measurements have been made onspectrofluorometer Shimadzu at excitation wavelength 384 nm and at 430to 510 nm emission wavelength range. Extinction coefficient at emission475 nm was determined as equal to 1.58×10⁶ M⁻¹. The specific amount ofprimary amino groups was 0.69 mmol/g.

EXAMPLE 32 Synthesis of Grafted Copolymer

Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol) by the same amount of Pluronic L61 (BASF Co.) andusing a molar ratio of activated Pluronic L61 to free amino groups ofpolyethyleneimine 0.3:1.0, there is obtained in 22% yield a graftedpolyethyleneimine copolymer in which 8% of free amino groups aresubstituted with Pluronic L61.

EXAMPLE 33 Synthesis of Grafted Copolymer

Following the procedure Of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic P85 and using amolar ratio of activated Pluronic P85 to free amino groups ofpolyethyleneimine 0.3:1.0 there is obtained in 70% yield a graftedpolyethyleneimine copolymer in which 11% of free amino groups ofpolyethyleneimine are substituted with Pluronic P85.

EXAMPLE 34 Synthesis of Grafted Copolymer

Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic L123 (BASF Co.)and using a molar ratio of activated Pluronic L123 to free amino groupsof polyethyleneimine 0.3:1.0 there is obtained in 30% yield a graftedpolyethyleneimine copolymer in which 9% of free amino groups aresubstituted with Pluronic L123.

EXAMPLE 35 Synthesis of Grafted Copolymer

Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic F38 (BASF Co.) andusing a molar ratio of activated Pluronic F38 to free amino groups ofpolyethyleneimine 0.3:1.0 there is obtained in 40% yield a graftedpolylysine copolymer in which 9% of free amino groups are substitutedwith Pluronic F38.

EXAMPLE 36 Synthesis of Multi-grafted Copolymer

Following the procedure of Example 28 but substituting polyethyleneimineby polyethyleneimine modified with Pluronic L123 (BASF Co.) obtained inExample 35 and using a molar ratio of activated poly(ethylene glycol) tofree amino groups of modified polyethyleneimine 0.4:1.0 there isobtained in 20% yield a grafted polyethyleneimine copolymer in which 9%of free amino groups are substituted with Pluronic L123 and 30% ofgroups are substituted with poly(ethylene glycol).

EXAMPLE 37 Complex with Oligonucleotide

A. Model phosphorothioate oligodeoxyribonucleotide PS-dT20 wassynthesized using ABI 291 DNA Synthesizer (Applied Biosystems, SanDiego, Calif.) following the standard protocols. After ammoniadeprotection the oligonucleotide was twice precipitated by ethanol andthen used without purification.

B. The complex formed between the PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer obtained inExample 28 was obtained by mixing the aqueous solutions of thesepolymers in 10 mM phosphate buffer, pH 7.4 so that the ratio of theprimary amino groups of the block copolymer to the phosphate charges ofthe PS-dT20 was 1.0. All solutions were prepared using double distilledwater and were filtered repeatedly through the Millipore membrane withpore size 0.22 μM.

C. The electrophoretic mobility (EPM) and the size of the particles ofthe complex synthesized in B were determine. The EPM measurements wereperformed at 25° C. with an electrical field strength of 15-18 V/cmusing “ZetaPlus” Zeta Potential Analyzer (Brookhaven Instrument Co.)with 15 mV solid state laser operated at a laser wavelength of 635 nm.The zeta-potential of the particles was calculated from the EPM valuesusing the Smoluchowski equation. Effective hydrodynamic diameter wasmeasured by photon correlation spectroscopy using the same instrumentequipped with the Multi Angle Option. The sizing measurements wereperformed at 25° C. at an angle of 90°. The zeta potential of thissample was close to zero, suggesting that particles were electroneutral.The average diameter of the particles was 35 nm.

EXAMPLE 38 Stability against Nuclease Digestion

100 μg of the complex formed between the PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer obtained inExample 39 was treated by 1 mg of snake venom phosphodiesterase(Phosphodiesterase I from Crotalus adamanteus, 0.024 units/mg, Sigma)for 2 and 18 hrs at 37° C. Reaction mixtures were analyzed by gelpermeation HPLC for digested PS-dT20. The digestion of the PS-dT20 inthis complex was less than 5%. In contrast, free PS-dT20 treated withthe same concentration of enzyme for the same time interval was digestedcompletely.

EXAMPLE 39 Accumulation of Oligonucleotide in Caco-2 Monolayers

A. A 5′-aminohexyl PS-dT20 oligonucleotide was synthesized using ABI 291DNA Synthesizer (Applied Biosystems, San Diego, Calif.) following thestandard protocols. After ammonia deprotection the oligonucleotide wastwice precipitated by ethanol and then used without purification.5′-Aminohexyl PS-dT20 was labeled by reaction with fluoresceinisothiocyanate (Sigma) following the manufacturer protocol.Fluorescein-labeled PS-oligonucleotide was separated from unreactedfluorophore using a Pharmacia PD-10 size exclusion.

B. The complex formed between the fluorescein-labeled PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer was synthesizedas described in Example 37 but using fluorescein-labeled PS-dT20 insteadof PS-dT20.

C. Caco-2 cells, originating from a human colorectal carcinoma (Fogh etal. J. Natl. Cancer Inst., 59:221-226, 1977) were kindly provided byBorchardt R. T. (The University of Kansas, Lawrence, Kans.). The cellswere maintained in Dulbecco's Modified Eagle's Medium (DMEM), containing10% heat-inactivated fetal bovine serum (FBS), 1% non-essential aminoacids, benzylpenicilin (100 U/ml) and streptomycin (10 ug/ml), in anatmosphere of 90% air and 10% CO₂ as described by Artursson (J. Pharm.Sci., 79:476-482, 1990). All tissue culture media were obtained fromGibco Life Technologies, Inc. (Grand Island, N.Y.). The cells were grownon collagen coated polycarbonate filter chamber inserts (Transwell,Costar Brand Tissue Culture Products, Contd.; pore size 0.4 um; diameter24.5 mm). 250,000 cells were added to each insert and cells of passagenumber 32-45 were used. The cells were fed every second day and wereallowed to grow and differentiate for up to 14 days before themonolayers were used in the following absorption experiments.

D. Caco-2 cell monolayers were preincubated for 30 min. at 37° C. withassay buffer, containing sodium chloride (122 mM), sodium bicarbonate(25 mM), glucose (10 mM), HEPES (10 mM), potassium chloride (3 mM),magnesium sulfate (1.2 mM), calcium chloride (1.4 mM) and potassiumphosphate dibasic (0.4 mM). After this, the assay buffer was removed andthe cells were exposed to 50 μM fluorescein-labeled PS-oligonucleotideor its complex in the assay buffer for 90 min at 37° C. After that thedye solutions were removed and cell monolayers were washed three timeswith ice-cold PBS. Cells were then solubilized in 1.0% Triton X-100 andaliquots (25 μl) were removed for determination of cellular fluorescenceusing a Shimadzu RF5000 spectrofluorometer at λex=488 nm, λem =520 nm.Samples were also taken for protein determination using the Pierce BCAmethod. The amounts of fluorescein-labeled PS-dT20 absorbed by the cellswas as follows:

Cellular accumulation of Sample oligonucleotide, nmol/mg protein Freefluorescein-labeled PS-dT20 0.14 ± 0.03 The complex  0.5 ± 0.01

This demonstrates that incorporation of polynucleotide in the complexwith the block copolymer increases cellular accumulation ofpolynucleotide by more than 3-times.

EXAMPLE 40 Transport of Oligonucleotide across Caco-2 Monolayers

A. The filter-grown Caco-2 monolayers were used for oligonucleotidepermeability studies after complete maturation, i.e., as from day 14after plating. Filters were gently detached from the wells and placed inSide-Bi-Side diffusion cells from Crown Bio Scientific, Inc.(Somerville, N.J.) maintained at 37° C.±0.1° C. This system is used asan in vivo model of human intestinal epithelium to evaluate oralbioavailability of drugs (Pauletti et al. Pharm. Res., 14:11-17, 1977).Cell monolayers were preincubated for 30 minutes at 37° C. with theassay buffer, containing 10% heat-inactivated fetal bovine serum (FBS),1% non-essential amino acids, benzylpenicilin (100 U/ml) andstreptomycini (10 ug/ml), added to both donor and receptor chambers (3ml). After preincubation, the assay buffer in the receptor container wasreplaced by the fresh one, while the assay buffer in the donor containerwas replaced by 50 μM fluorescein-labeled PS-oligonucleotide or itscomplex in the assay buffer. To account for the integrity of themonolayers the rhodamine 123 solutions in the donor container alsocontained H³-labeled manitol, a paracellular marker (Dawson, J. MembraneBiol., 77: 213-233, 1977) obtained from DuPont Corp. (Boston, Mass.). At120 min., the solutions in the receptor chamber were removed fordetermination of fluorescein-labeled PS-oligonucleotide using a ShimadzuRF5000 fluorescent spectrophotometer (λex=488 nm, λem=520 nm) andH³-manitol determination using a liquid scintillation counter (HewlettPackard Instruments). Immediately after collecting the solutions in thereceptor chamber 3 ml of fresh assay buffer was added to this chamber.The transport of fluorescein-labeled PS-oligonucleotide (or manitol)across Caco-2 cell monolayers was expressed as a percentage of the totalfluorescein-labeled PS-oligonucleotide (or manitol) accumulated in thereceptor chamber to the initial amounts of fluorescein-labeledPS-oligonucleotide (or manitol) in the donor chamber. A minimum of threedifferent membranes was studied for each drug composition at multipletime points for each membrane. The results were as follows:

Sample PS-dT20 transport, % Manitol transport, % Freefluorescein-labeled 0.001 ± 0.0005 4.0 ± 0.1  PS-dT20 The complex 0.075± 0.005  4.2 ± 0.02

This demonstrates that incorporation of polynucleotide in the complexwith the block copolymer increases transport of this polynucleotideacross Caco-2 monolayers by more than 7-times while the transport ofparacellular marker is not affected.

EXAMPLE 41 Synthesis of Polyvinylpyrrolidone-Polyethyleneimine Conjugate

A. Carboxyterminated polyvinylpyrrolidone was obtained using the methoddescribed by Torchilin et al. (J. Pharm. Sci. 84:1049, 1995).Polyvinylpyrrolidone was synthesized by chain transfer free radicalpolymerization of 50% wt. N-vinylpyrrolidone (Sigma) inisopropoxyethanol with 1% wt. 2,2′-azoisobutyronitrile (Sigma) as theinitiator. The MW of the polymer obtained was about 6,000 as determinedby viscosimetry and gel-permeation chromatography on Sephadex G25. Theterminal OH group of polyvinylpyrrolidone was converted into a COOHgroup by activating the OH group with 4-nitrophenyl chlorophormate andsubsequently coupling it with glycine as described by Sartore et al., J.Bioact. Compat. Polym. 9:411 (1994).

B. Six grams (1 mmole) of carboxyterminated polyvinylpyrrolidoneobtained in Part A dissolved in 30 ml of dry dioxane was treated with0.2 g of N,N′-dicyclohexylcarbodiimide for 3 hours at room temperatureand then reacted with 2.5 g (0.1 mmole) polyethyleneimine, Mw. 25,000(Aldrich Co.) for 15 hours at room temperature. The reaction mixture wasthen dialyzed against water for 3 days using Spectra/Por membranes andthen purified by high-pressure liquid to chromatography using SilasorbC18 column in the gradient of acetonitrile. Three (3) g ofpolyvinylpyrrolidone-polyethyleneimine conjugate was obtained.

EXAMPLE 42 Synthesis of Polyaryloylmorpholine-PolyethyleneimineConjugate

A. Carboxyterminated polyaryloylmorpholine was obtained using the methoddescribed by Torchilin et al. (J. Pharm. Sci., 84:1049, 1995). Themonomer was synthesized by acylation of morpholine (Aldrich) withacryloyl chloride (Aldrich), which was then polymerized in water with 1%wt. 2,2′-azoisobutyronitrile (Sigma) as the initiator and2-mercaptoacetic acid as chain transfer reagent (Ranucci et la.,Macromol. Chem. Phys., 195:3469, 1994). The molecular weight (MW) of thepolymer obtained was ca. 8,000 as determined by viscosimetry andgel-permeation chromatography on Sephadex G25.

B. Following the procedure of Example 40 but substituting 16 g ofcarboxyterminated polyaryloylmorpholine for 6 g of carboxyterminatedpolyvinylpyrrolidone in Part B there was obtained 4 g ofpolyaryloylmorpholine-polyethyleneimine conjugate.

EXAMPLE 43 Synthesis of Polyvinylyrrolidone-Polyethyleneimine Conjugate

A. Carboxyterminated polyacrylamide was obtained using the methoddescribed by Torchilin et al. (Biochim. Biophys. Acta., 1195:181, 1994).Ten percent by weight of acrylamide (Aldrich) was polymerized in dioxanewith 1% wt. 2,2′-azoisobutyronitrile (Sigma) as initiator and2-mercaptoacetic acid as chain transfer reagent. The MW of the polymerobtained was ca. 5,000 as determined by viscosimetry and gel-permeationchromatography on Sephadex G25.

B. Following the procedure of Example 40 but substituting 25 g ofcarboxyterminated polyacrylamide for 6 g of carboxyterminatedpolyvinylpyrrolidone in Part B, 10 g ofpolyvinylpyrrolidone-polyethyleneimine conjugate was obtained.

EXAMPLE 44 Synthesis of Polyacrylamide-Polyethyleneimine Conjugate

Following the procedure of Example 42 but substituting vinyltriazolemonomer for acrylamide monomer in Part A, 9.2 g ofpolyacrylamide-polyethyleneimine conjugate was obtained.

EXAMPLE 45 Synthesis of Polyvinylalcohol-graft-PolyethyleneimineCopolymer

20 g of polyvinylalcohol, mw. 100,000 (Aldrich) was activated by 2.5 gof 1,1′-carbonyldiimidazole in 30 ml of anhydrous acetonitrile for 4 hrsat room temperature. The solvent was evaporated in vacuo, and theresidue redissolved in water and dialyzed through Membra-Cel MD-25-03.5membrane against water. Desalted solution was concentrated in vacuo andused in a reaction with 5 g of polyethyleneinimine, mw. 2000, inmethanol-water solution for 30 hrs at room temperature. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia). 16.5 g ofpolyvinylalcohol-graft-polyethyleneimine copolymer was obtained.

EXAMPLE 46 Synthesis of monoamino-poly(ethylene glycol)

A. Poly(ethylene glycol), 1 MW 8,000, obtained from Aldrich (St Louis,Mo.) was modified by one terminal hydroxyl group using with4,4′-dimethoxytrityl (DMT) chloride (Sigma, St Louis, Mo.). Briefly, 16g (2 mmoles) of PEG were dried by coevaporation in vacuo with anhydrouspyridine (3×50 ml) and dissolved in 100 ml of the solvent. 0.34 g (1mmole) of 4,4′-dimethoxytrityl chloride in 10 ml of anhydrous pyridinewas then added to this solution dropwise for 30 min and the mixture wasleft under stirring overnight at 25° C. Monotritylated polymer waseseparated from fast moving bis-substituted by-products and non-reactedinitial compounds by preparative column chromatography on Silicagel(Selecto Scientific, Norcross, Ga.), particle size 32-63 μm, using astepwise elution by methanol in dichloromethane. Fractions containingmono DMT-substituted poly(ethylene glycol) (DMT-PEG) were collected andsolvents were removed in vacuo. DMT-PEQ was obtained with 40% yield.

B. To prepare the monoamino-poly(ethylene glycol) (N-PEG), 1.5 g ofDMT-PEG (0.18 mmol) was coevaporated twice in vacuo with anhydrousacetonitrile (10 ml) and treated by an excess of 1,1′-carbonyin bound 34μg of biotin-poly(ethylene glycol)-polyethylenymer comprising apolyether segment and a polycation segment. In yet another aspect, theinvention provides polynucleotides 10 that have been covalently modifiedat their 5′ or 3′ end to attach a polyether polymer segment.

What is claimed is:
 1. A method for delivering a polynucleotide to acell comprising administering to the cell an effective amount of acomposition comprising noncovalent complex of said polynucleotide and apolymer comprising a plurality of covalently bound segments comprising:(a) at least one polycationic homopolymer or copolymer having at leastthree cationic sites, or a protonated or quaternary form thereof, saidpolycationic homopolymer or copolymer comprising at least one memberselected from the group consisting of (i) a tertiary amino monomer ofthe formula:

 and (ii) a secondary amino monomer of the formula:

 in which: each of R¹, R⁵, R⁶, and R⁸, independently of the others, ishydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer;each of R², R³, and R⁷, taken independently of the others, is the sameor different straight or branched chain alkanediyl group of the formula:

in which z has a value of from 2 to 8; and R⁴ is hydrogen; and (b) atleast one water-soluble nonionic polymer segment.
 2. The method of claim1 wherein the water-soluble nonionic polymer segment is a homopolymer orcopolymer of at least one monomer selected from the group consisting ofacrylamide, glycerol, vinyl alcohol, vinylpyrrolidone, vinylpyridine,vinylpyridine N-oxide, oxazoline, and acryloyl morpholine.
 3. The methodof claim 1 wherein said water-soluble nonionic polymer segment comprisesat least one straight or branched chained polyether segment which is:(i) a homopolymer of a first alkyleneoxy monomer —OC_(n)H_(2n)— in whichn has a value of 2 or 3; or (ii) a copolymer or block copolymer of saidfirst alkyleneoxy monomer and a second different alkyleneoxy monomer—OC_(m)H_(2m)— in which m has a value of from 2 to 4, said polyethersegment having from about 5 to about 400 monomeric units.
 4. The methodof claim 1 wherein the composition comprises a surfactant.
 5. The methodof claim 4 wherein the surfactant is cationic, nonionic, orzwitterionic.
 6. The method of claim 1 wherein the polynucleotide is aviral polynucleotide.
 7. The method of claim 1 wherein the compositioncomprises a targeting molecule.
 8. The method of claim 7 wherein thetargeting molecule is a peptide or polypeptide.
 9. The method of claim1, wherein the polynucleotide is DNA.
 10. The method of claim 1 whereinthe composition comprises a ligand.
 11. The method of claim 1 whereinthe composition comprises a receptor.
 12. The method of claim 1 whereinthe water-soluble nonionic polymer is of the formula:

in which m has a value of from 3 to about 10,000.
 13. The method ofclaim 1 wherein the polynucleotide is delivered ex vivo to a cell.